CN107847763A - Targeting selection is suitable for the patient with cortex chalone derivatives for treatment - Google Patents

Abstract

A method for targeted selection and treatment of tumors or cancer patients, comprising (i) determining whether the patient has RUNX1 pathway impairment; if so, (ii) optionally administering an effective amount of Cortistatin or a pharmaceutically acceptable salt or oxide thereof.

Description

Targeted selection for patients treated with corticostatin derivatives

related application

This application is related to and claims Provisional U.S. Application No. 62/158,936, filed May 8, 2015, Provisional U.S. Application No. 62/187,656, filed July 1, 2015, and Provisional U.S. Application No. 62/187,656, filed February 22, 2016 Benefit of No. 62/298,352. The entire contents of these provisional applications are hereby incorporated by reference for all purposes.

Background technique

U.S. Patent 9,127,019, entitled "Corticostatin Analogs and Synthesis Thereof," filed by Flyer et al. and assigned to the Dean and Fellows of Harvard University, describes the synthesis of cortistatins A, J, K, and L having the general formula I Analogs or salts thereof and their synthesis, wherein R1, R2, R3, R4, n and m are as described therein.

The '019 patent discloses that such compounds are anti-angiogenic and can be used in the treatment of proliferative diseases.

WO2015/100420 entitled "Cortistatin Analogs and Synthesis and Uses Thereof", filed by Shair et al. and also assigned to the Dean and Fellows of Harvard University, describes other analogs and methods of cortistatin and includes the described Compositions of cortistatin analogues for use in the treatment of proliferative diseases such as cancer, especially hematopoietic cell carcinomas, such as epistaxis, multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms, acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML) and primary myelofibrosis (PMF). More generally, the '420 application describes methods of treating disorders associated with CDK8 and/or CDK19 kinase activity comprising administering an effective amount of a disclosed compound, or a pharmaceutically acceptable salt, quaternary amine or N-oxide thereof. CDK8 and its regulatory subunit cyclin C are components of the RNA polymerase II halogenase complex, which phosphorylates the carboxy-terminus of the largest subunit of RNA polymerase II. CDK8 regulates transcription by targeting the CDK7/cyclin H subunit of the universal transcription factor TFIIH

Additional synthesis and biology of cortistatin A and cortistatin A analogues have been described in Chiu et al. Chemistry (2015), 21: 14287-14291, entitled "Formal Total Synthesis of (+)-Cortistatin A and J" ; Valente et al. Current HIV Research (2015), 13:64-79, titled "Didehydro-Cortistatin A Inhibits HIV-1 Tat Mediated Neuroinflammation and Prevents Potentiation of Cocaine Reward in Tat TransgeniCMice"; Motomasa et al., Chemical & Pharma. Bulletin (2013 ), 61:1024-1029, titled "Synthetic Studies of Cortistatin A Analog from the CD-ringFragment of Vitamin D2"; Valente et al., Cell Host & Microbe (2012), 12:97-108 titled "An Analog of the Natural SteroidalAlkaloid Cortistatin A Potently Suppress Tat-dependent HIV Transcription”; Motomasa et al., ACS Med. Chem. Lett. (2012), 3: 673-677 titled “Creation of Readily Accessible and Orally Active Analog of Cortistatin A”; Danishefsky et al. al., Tetrahedron (2011) 67:10249-10260 titled "Synthetic Studies Toward(+)-Cortistatin A"; Motomasa et al., Heterocycles (2011), 83:1535-1552, titled "Synthetic Study of Carbocyclic Core of Cortistatin A" A, an Anti-angiogenic Steroidal Alkaloid from Marine Sponge”; Motomasa et al., Org. Lett. (2011), 13:3514-3517, titled "Stereoselective Synthesis of Core Structure of Cortistatin A"; Baran et al., JACS (2011), 133:8014-8027, titled "Scalable Synthesis of Cortistatins A and Related Structures"; Hirama et al., JOC (2011), 76: 2408-2425, titled "Total Synthesis of Cortistatins A and J"; Zhai et al., Org. Lett. (2010), 22:5135-5137, entitled "Concise Synthesis of the Oxapentacyclic Core of Cortistatin A"; Stoltz et al., Org. Biomol.Chem. (2010), 13:2915-2917, entitled "Efforts Toward Rapid Construction of the Cortistatin A Carbocyclic Core via Enyne-ene Metathesis”; Sarpong et al., Tetrahedron (2010), 66: 4696-4700, titled “Formal Total Synthesis of (±)-Cortistatin A”; Nicolaou et al., Angewandte Chemie (2009), 48:8952-8957, described in the title "Cortistatin A is a High-Affinity Ligand of Protein Kinases ROCK, CDK8, and CDK11".

US Patent Application Publication US2013/0217014 and PCT Application WO2013/122609, entitled "Methods of Using CDK8 Antagonists", filed by Firestein et al. and assigned to Genentech, describe the use of CDK8 antagonists against various cancers. CDK8 has a conserved function in transcription as part of the Mediator complex, as described therein, as Taatjes, D.J., Trends Biochem Sci 35, 315-322 (2010); and Conaway, R.C. and Conaway, J.W., CurROpin Genet Dev 21, 225- 230 (2011). CDK8 has also been reported for colon cancer (Firestein R. et al., Nature 455:547-51 (2008); Morris E.J. et al., Nature 455:552-6 (2008); Starr T.K. et al., Science 323:1747-50 (2009) ) and oncogenes in melanoma (Kapoor A. et al., Nature 468:1105-9 (2010)). CDK8 is upregulated and amplified in a subset of human colon tumors and is known to transform immortalized cells and is required for colon cancer proliferation in vitro. Likewise, CDK8 was also found to be overexpressed in melanoma and is essential for proliferation. Kapoor, A. et al., Nature 468, 1105-1109 (2010). CDK8 has been shown to regulate several signaling pathways that are key regulators of ES pluripotency and cancer. CDK8 activates the Wnt pathway by promoting the expression of β-catenin target genes (Firestein, R. et al., Nature 455, 547-551 (2008)) or by inhibiting E2F1, a potent inhibitor of β-catenin transcriptional activity. Morris, E.J. et al., Nature 455, 552-556 (2008). CDK8 promotes the expression of Notch target genes by phosphorylating the Notch intracellular domain, activating the Notch enhancer complex at the target genes. Frye RC. J. et al., Mol Cell 16:509-20 (2004).

It is well known that tumors and cancers can be heterogeneous even within narrow categories. See, eg, Meacham et al., TumoRheterogeneity and cancerRcell plasticity, Nature Vol. 501, 328-337 (September 19, 2013). Because specific tumor types can be caused by a range of genetic abnormalities that can, as a result, express or repress key proteins, resulting in a range of phenotypes, not all tumors or cancers within a narrow range will respond to the same drug treatments. Even with the most active oncology drugs, there are expected to be responders and non-responders.

Therefore, it would be advantageous to provide a method of determining which tumors and cancer cells respond best to cortistatin treatment.

It would also be advantageous to enable targeted selection of patients with tumors or cancers that respond optimally to cortistatin therapy.

It would be further advantageous to enable targeted selection of patients with tumors or cancers with the best response to cortistatin therapy, wherein the tumors or cancers are of hematopoietic lineage.

Contents of the invention

In a first embodiment of the present invention, cortistatin has been found to be particularly useful for the treatment of tumors and cancers with impaired Runt-related transcription factor 1 (RUNX1) transcriptional programs. Based on this finding, a method for targeted selection and treatment of patients more likely to respond to cortistatin therapy is proposed, which includes (i) determining whether the patient has RUNX1 pathway impairment; if so, (ii) administering an effective amount Cortistatin derivatives, including, for example, cortistatin derivatives or pharmaceutically acceptable salts and/or compositions thereof as described herein. For example, RUNX1 impairment may result from a RUNX1 point mutation, a chromosomal translocation involving the RUNX1 gene, or a mutation that causes RUNX1 protein instability or increased degradation.

In one aspect, there is provided a method for treating RUNX1 impaired tumors or cancers by administering an effective amount of cortistatin in a manner and at a dose to produce sufficient upregulation of a protein normally transcribed by RUNX1 to render the cells more normal , less virulent, more mature or prevents growth or apoptosis causing differentiation of said tumor or cancer.

For example, a method of predicting the response of a patient with a tumor or cancer to treatment with cortistatin, comprising the steps of: obtaining a sample of the tumor or cancer from the patient, and detecting one or more biomarkers in the biological sample from the patient The expression level or amount of expression, wherein the biomarkers are selected from the group consisting of ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPα, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10, E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4, IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK, PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3, ZBTB16, and ZCCHC5; then determine whether the expression level or amount assessed is outside the range of the corresponding normal cells, e.g., higher or lower than that found in the corresponding normal cells range, or above or below a certain amount associated with an increase or decrease in clinical benefit to the patient; then optionally with an effective amount of cortistatin or a pharmaceutically acceptable salt or oxide thereof, optionally at The patient is treated in its pharmaceutically acceptable composition. In an alternative embodiment, the method comprises comparing the expression of the selected gene to the expression of the same gene in a control sample comprising a representative number of cortistatin responsive patients or Predictive animal models and representative numbers of patients with no or poor response to cortistatin to determine whether patients tend to respond to cortistatin therapy.

Also provided are kits for determining whether a patient will successfully respond to cortistatin therapy, which may include a probe that anneals to a biomarker or a polynucleotide in combination with a biomarker under stringent conditions or binds to a biomarker protein antibodies. The kit may include primers for amplifying DNA complementary to the RNA specifically encoded by the gene, and optionally a thermostable DNA polymerase. In one embodiment, the primers hybridize under standard stringent conditions to the RNA encoded by the selected gene or its complement.

The selected biomarker may in one aspect be one or a combination of GATA1, GATA2, C/EBPa, FLI1, FOG1, ETS1, PU.1, RUNX1, and CBFα. Alternatively, selected biomarkers are BCL2, CCNA1, CD44, C/EBPa, CBFβ, CSF1, CXCL10, CXCR4, ETS1, ETS2, FLI1, FOG1, FCER1A, GATA1, GATA2, GFI1B, HEB, IRF1, IRF8, JAG1 , LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1 and TNF or a combination thereof. In various embodiments, the selected biomarker is constitutive STAT1-pS727, WT1 mutation, TET2 mutation, IDH1 mutation, IDH2 mutation, MLL rearrangement, C/EBPa mutation, CBFβ rearrangement, PU.1 mutation, One or a combination of GATA1 or 2 mutations, ERG translocations, TLX1 overexpression, and TLX3 activation.

Also provided is a method for targeted selection and treatment of patients likely to respond to cortistatin therapy, the method comprising (i) determining whether the patient has a disease selected from the group consisting of ER-positive, VHL loss-of-function mutations (VHL-negative), HER2 overexpression , EGFR mutation, MET mutation, a biomarker for neuroblastoma, EIVS-FLI1, STAT1-pS727, STAT1, or one of the biomarkers for inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2 or in combination, and if so, (ii) administering an effective amount of a cortistatin derivative (including, for example, that described herein) a pharmaceutically acceptable salt, oxide, and/or composition thereof.

In another aspect, at least two, three, four, five or more of any of the biomarkers described herein are used for target selection with an effective amount of cortistatin or a salt thereof, N-oxide and/or a pharmaceutically acceptable composition thereof for treating tumor or cancer.

Treatable non-limiting hematopoietic lineage tumors or cancers may be selected, for example, from: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), childhood B-ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related lymphoma, chronic myeloproliferative disorder, primary central nervous system lymphoma, T-cell lymphoma, hair cell leukemia, and multiple myeloma (MM).

The invention includes the treatment of cells that are precursors of hematopoietic neoplasms or cancers, such as those found in myelodysplastic syndrome (MDS).

The tumor or cancer may also be a tumor or cancer of a non-hematopoietic lineage, such as breast cancer, ovarian cancer, endometrial cancer, squamous cell carcinoma, angiosarcoma, colon cancer, gastrointestinal tumors, metastatic-prone solid tumors, clear cell carcinoma, renal Cell carcinoma or cancer of the esophagus.

Accordingly, the present disclosure provides methods for overcoming RUNX1 inactivating mutations based on the surprising discovery that inhibition of CDK8 and CDK19 with cortistatins, including but not limited to those disclosed herein, by causing Upregulation of RUNX1 target genes reverses the effects of RUNX1-inactivating mutations. Because of this surprising effect, cortistatin may be useful in the treatment of malignancies associated with inactivating RUNX1 mutations, for example by administering CDK8/19 inhibitors and/or cortistatin or its corticostatin analogues to patients with Individuals with cancer associated with RUNX1 inactivating mutations.

For example, cortistatin has been found to potently inhibit the proliferation of many AML cell lines at a ₅₀ % maximal growth inhibitory concentration (GI50) of less than 10 nM. Cell line sensitivity is consistent with RUNX1 transcriptional program dependence. Sensitive cell lines include cells that directly inhibit the transcription of RUNX1 or its target genes (SKNO-1, ME-1, MOLM-14, MV4; 11) and those with truncated GATA-1 proteins, GATA-1s (CMK-86 and MEG -01) of the fusion of megakaryocytic leukemia cell lines. Unlike megakaryocytes, RUNX1 expression declines rapidly during erythroid terminal differentiation, consistent with cortistatin insensitivity of erythroleukemic cell lines.

Cortistatin upregulates RUNX1 target genes, including CEBPA, IRF8, and NFE2. By Genome Enrichment Analysis (GSEA), it was determined that (i) cortistatin upregulates SET-2, MOLM-14, and MV4;11 genes in cell lines, which are repressed by the expression of RUNX1-RUNX1T1 in hematopoietic stem cells; (ii) cortistatin Upregulation of MOLM-14 and MV4 by cortistatin; 11 genes in the cell line whose expression was reduced in the Kasumi-1 AML cell line after siRNA-mediated knockdown of RUNX1; and (iii) upregulation of cortistatin in MOLM-14 cells gene whose expression was increased in Kasumi-1 cells after siRNA-mediated knockdown of RUNX1. RUNX1 is recruited to loci upregulated by cortistatin treatment.

Aspects of the present disclosure provide methods of diagnosing cancer in an individual that is sensitive to treatment with a CDK8/19 inhibitor and/or cortistatin or a cortistatin analog, the method comprising (a) determining whether the individual has exhibited a cancer exhibiting impaired RUNX1 activity; and (b) identifying said individual as having a cancer sensitive to said compound treatment if said individual is determined to carry a cancer exhibiting impaired RUNX1 activity. In some embodiments, the method further comprises administering to the individual a CDK8/19 inhibitor and/or cortistatin or a cortistatin analog in an amount effective to treat the cancer.

In some embodiments of the methods of diagnosis and treatment provided herein, the cancer is a hematological cancer associated with an inactivating mutation of RUNX1. In some embodiments, the cancer is leukemia, such as acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic myelomonocytic Cellular leukemia (CMML). In some embodiments, the acute lymphoblastic leukemia is T-cell acute lymphoblastic leukemia, childhood precursor B-ALL, or B-cell acute lymphoblastic leukemia. In some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, or squamous cell carcinoma.

Aspects of the present disclosure provide pharmaceutical compositions and kits comprising cortistatin or a pharmaceutically acceptable salt thereof, a quaternary ammonium salt or an N-oxide thereof, for example for use in the treatment of cancers exhibiting impaired RUNX1 activity A drug wherein cortistatin has the formula (A-1), (A-1′), (A-1″), (A-2′), (A-2″), (A-3′), (A-3″), (D1′), (D1″), (D2′), (D2″), (E1′), (E1″), (E2′), (E2″), (G1′ ) or (G1") or a pharmaceutically acceptable salt thereof.

The above summary is intended to illustrate, in a non-limiting manner, some embodiments, advantages, features and uses of the technology disclosed herein. Other embodiments, advantages, features and uses of the technology disclosed herein will be apparent from the detailed description, drawings, implementations and claims.

Description of drawings

Figure 1 shows the relationship between the Mediator complex and various transcriptional regulators. CDK8 and CDK19 associate with Mediator and regulate transcription. RUNX1 binds to enhancer elements, including super-enhancers, and functions with transcription factors including, but not limited to, TAL1, C/EBPa, CBFβ, FLI1, ETS1, FOG1, GATA1, and PU.1. Many of these transcription factors have been found to be mutated in some AML patients, including RUNX1, C/EBPa, and GATA1. Treatment with the CDK8/19 inhibitor cortistatin A increased the expression of RUNX1 target genes and super-enhancer-associated genes. Many RUNX1 target genes whose expression increased after cortistatin A treatment were also super-enhancer-associated genes.

Figure 2 is a gene enrichment analysis of RUNX1 target genes in AML, plotted against their interaction with cortistatin A. Cortistatin A upregulates RUNX1 target genes in AML, and Genome Enrichment Analysis (GSEA) profiles revealed that 3h 25nM cortistatin A treatment upregulated genes in MOLM-14 cells that were in RUNX1-RUNX1T1 (also known as AML1- ETO) was upregulated in Kasumi-1 cells after knockdown.

Figure 3 is a bar graph of the percentage of cells with the megakaryocyte markers CD41 and CD61 in the presence of vehicle, 50 nM cortistatin A, or 50 ng/mL PMA. Treatment with the CDK8/19 inhibitor cortistatin A induced differentiation of SET-2 cells as measured by increases in the megakaryocyte markers CD41 and CD61. CD41 and CD61 on SET-2 cells (vehicle vs. CA, p=0.04 and 0.005, respectively, two-tailed t-test) after 3 days of the indicated treatments (mean ± standard error, n = 3).

Figure 4 is a graph of theoretical cell number versus days of cortistatin A treatment. Treatment with the CDK8/19 inhibitor cortistatin A inhibits the proliferation of SET-2 cells. Plots of CA cell numbers over time (mean±SE, n=3) show dose-dependent effects.

Figure 5 is a graph of synergy in inhibiting the proliferation of MPN/AML cell lines SET-2 and UKE-1, where the combination index is plotted against the combination of the CDK8/19 inhibitor cortistatin A (CA) and the JAK1/2 inhibitor Ruxolib Graph of the ratio of ruxolitinib. This figure shows that CDK8/19 inhibition works synergistically with JAK1/2 inhibition. Synergy was determined using the method of Chou-Talalay (CalcuSyn).

Figure 6 is a graph of spleen weights of mice with AML under different doses of cortistatin A. Cortistatin A treatment prevented increased spleen weight in female NOD-SCID-IL2Rcγ ^null (NSG) mice with disseminated MV4;11-mCLP leukemia that had been treated with corticosteroid A once daily by IP administration for 15 days. Points represent values for individual mice after cessation of cortistatin A treatment for an additional 15 days and 37 days after tail vein injection of 2 million MV4;11-mCLP cells. Dashed lines represent ranges within 1 standard deviation of the mean for relative healthy 8-week-old female NOD-SCID mice and were obtained from the Mouse Phenotype Database 22903 (The Jackson Laboratory).

Figure 7A is a graph of percent kinase activity remaining at 600 nM cortistatin A versus 294 recombinant kinases. Cortistatin A selectively inhibits CDK8/19 as measured by kinase assay assay (Wild Type Analyzer, ProQinase). These genome-wide profiling studies revealed that the CDK8/19 inhibitor cortistatin A is highly selective for CDK8/19.

Figure 7B is a graph of % inhibition of native kinase activity at 1,000 nM cortistatin A. Cortistatin A selectively inhibits CDK8/19 as measured by a native kinase assay (KiNativ, ActivX Biosciences). These genome-wide profiling studies revealed that the CDK8/19 inhibitor cortistatin A is highly selective for CDK8/19.

Figure 8 is a graph of percent kinase activity versus concentration of cortistatin A on a logarithmic scale. This figure shows that cortistatin A potently inhibits CDK8/cyclin C in vitro.

Figure 9 is a graph of % growth versus cortistatin A concentration (nM, log scale) for WT and CDK8 and CDK19 mutants. Drug-resistant alleles demonstrate that CDK8/19 kinase activity is required for AML cell growth. This suggests that the CDK8/19 inhibitor cortistatin A inhibits the proliferation of MOLM-14 cells by inhibiting CDK8/19. Mutation of tryptophan 105 (W105) in CDK8 and CDK19 confers cortistatin A resistance to CDK8 and CDK19. Thus, MOLM-14 cells were able to proliferate in the presence of cortistatin A upon expression of CDK8W105M or CDK19W105M.

Figure 10: Analysis of MV4;11 AML mice at day 30 shows that treatment with the CDK8/19 inhibitor cortistatin A has fewer leukemic cells in the lungs as measured by hematoxylin and eosin staining.

Figure 11 is a gene enrichment analysis of genes with increased RUNX1 density, plotted against their interaction with cortistatin A. Cortistatin A upregulates SET-2, MOLM-14, and MV4;11 genes in cell lines, which are repressed by RUNX1-RUNX1T1 expression in hematopoietic stem cells (HSCs).

Figure 12 is a Western blot showing that Cas9 can also be used to knock out the endogenous gene BCL2L11. sgRNA #1 and #5 targeting only the EL and L isoforms strongly reduced the gene product Bim targeting all three isoforms compared to non-targeting controls. Although less efficient, sgRNA #4 targets all three isoforms. sgRNAs #2 and #3 target introns and do not reduce Bim.

Figure 13 shows that in cells expressing Cas9 and sgRNA #1 or #3 against ZsGREEN, green fluorescence was reduced to levels similar to control non-fluorescent cells. Sequencing of the ZsGREEN locus in cells expressing sgRNA#1 revealed an indel at the expected cleavage site.

Figure 14 is a screening workflow in which (A) blasticidin is used to select for stable expression of Cas9 in a cell line of interest, and then (B) lentiviral plasmids encoding sgRNAs targeting approximately 18,000 human genes and Puromycin Primers were introduced into the library for 7 days, after which (C) Day 0 at the start of screening, cells were treated with vehicle or CA for 14 days. (D) The distribution of each sgRNA was determined in the day 0 reference, day 14 vehicle-treated, and day 14 CA-treated populations. sgRNAs that were significantly enriched or depleted in the CA-treated group were representative biomarkers of CDK8/19 inhibition.

Figures 15A, 15B and 15C are graphs of growth levels measured in % for various cell lines in the presence of 100 nM cortistatin A.

Detailed ways

The present invention at least includes the following features:

A) A method of targeting and treating a patient with a tumor or cancer prone to respond to cortistatin therapy comprising (i) determining whether the patient has impaired RUNX1 pathway; if so, (ii) administering an effective amount of corticostatin Derivatives of cortistatin, including, for example, derivatives of cortistatin as described herein, or pharmaceutically acceptable salts, oxides and/or compositions thereof.

B) A method of treating RUNX1-impaired tumors or cancers by administering an effective amount of cortistatin in a manner and at a dose that produces sufficient upregulation of the protein normally transcribed by RUNX1 to render the cells more normal, less virulent, and more mature And prevent the differentiation of tumors or cancers in a way that prevents growth or apoptosis.

C) The method of A) or B), comprising using a kit to determine whether a patient will successfully respond to cortistatin therapy, said kit comprising a polynucleotide in combination with a biomarker or biomarker under stringent conditions Annealed probes or antibodies that bind biomarker proteins.

D) a method for predicting the response of a patient suffering from a tumor or cancer to treatment with cortistatin comprising the steps of:

a. Obtaining a tumor or cancer sample from a patient;

b. Determining the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPα, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10, E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4, IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK, PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3, ZBTB16, and ZCCHC5;

c. Determine whether the expression level or amount assessed in b is outside the range of the corresponding normal cells, e.g., above or below the range found in the corresponding normal cells, or above or below the increase with the patient or a certain amount associated with reduced clinical benefit; and

d. optionally treating the patient with an effective amount of cortistatin, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

E) A method of selecting a tumor or cancer patient suitable for treatment with cortistatin, said method comprising:

a. obtaining a patient tumor or cancer sample;

b. Detecting the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of: ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPα, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10, E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4, IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK, PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3, ZBTB16, and ZCCHC5;

c. Comparing the expression determined in step b with the expression of the same gene in a control sample comprising a representative number of cortistatin-sensitive responding patients or predictive animal models and a representative number of patients with no or poor response to cortistatin; and

d. If it is determined that the patient is prone to respond to treatment, administering an effective amount of cortistatin, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

F) The methods of A) to E), comprising a kit for assessing the expression level of a selected gene for diagnosing impaired RUNX1 pathway, primers for amplifying DNA complementary to RNA specifically encoded by the gene, and any Thermostable DNA polymerase of choice.

G) The method of F), wherein each primer hybridizes to the RNA encoded by the selected gene or its complement under standard stringent conditions.

H) The method of A) to G), wherein the selected biomarker is one or a combination of GATA1, GATA2, C/EBPa, FLI1, FOG1, ETS1, PU.1, RUNX1 and CBFα.

I) The methods of A) to G), wherein the selected biomarkers are BCL2, CCNA1, CD44, C/EBPa, CBFβ, CSF1, CXCL10, CXCR4, ETS1, ETS2, FLI1, FOG1, FCER1A, GATA1, GATA2, One or a combination of GFI1B, HEB, IRF1, IRF8, JAG1, LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1 and TNF.

J) The method of A) to G), wherein the selected biomarker is constitutive STAT1-pS727, WT1 mutation, TET2 mutation, IDH1 mutation, IDH2 mutation, MLL rearrangement, C/EBPa mutation, CBFβ rearrangement, PU .1 mutation, GATA1 or 2 mutation, ERG translocation, TLX1 overexpression, and TLX3 activation, or a combination thereof.

K) The method of A) to J), comprising using at least two biomarkers independently selected from the lists in D), H), I) and J).

L) The method of A) to J), comprising using at least three biomarkers independently selected from the lists in D), H), I) and J).

M) The method of A) to J), comprising using at least four biomarkers independently selected from the lists in D), H), I) and J).

N) The methods of A) to M), wherein the tumor or cancer is a tumor or cancer of the hematopoietic lineage.

O) The method of N), wherein the tumor or cancer of the hematopoietic lineage is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), childhood B-ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related lymphoma, chronic myeloproliferative disease, primary central nervous system lymphoma, T-cell lymphoma, hair cell leukemia, and multiple myeloma (MM), or where the cells are hematopoietic neoplasms or cancers such as myeloproliferative Precursor cells of dysmorphic syndrome (MDS).

P) The methods of A) to M), wherein the tumor or cancer is a tumor or cancer of non-hematopoietic lineage.

Q) The method of P), wherein the tumor or cancer is breast cancer, ovarian cancer, endometrial cancer, squamous cell carcinoma, angiosarcoma, colon cancer, gastrointestinal tumors, metastatic-prone solid tumors, clear cell carcinoma, renal cell carcinoma cancer or esophageal cancer.

R) The methods of A) to Q), wherein the cortistatin administered to the patient is selected from the group consisting of formulas (A-1), (A-1′), (A-1″), (A-2′), ( A-2″), (A-3′), (A-3″), (D1′), (D1″), (D2′), (D2″), (E1′), (E1″), Compounds of (E2'), (E2"), (G1') or (G1").

S) The methods of A) to Q), wherein the cortistatin administered to the patient is:

T) The methods of A) to Q), wherein the cortistatin administered to the patient is native cortistatin.

U) The methods of A) to Q), wherein the cortistatin administered to the patient is selected from known cortistatin derivatives.

V) The methods of A) to V), wherein the RUNX1 impairment is the result of a RUNX1 point mutation, a chromosomal translocation involving the RUNX1 gene, or a mutation leading to increased destabilization or degradation of the RUNX1 protein.

W) The methods of A) to V), wherein the impairment of the RUNX1 transcription factor results in reduced expression of genes under the control of RUNX1.

X) A method for targeted selection and treatment of patients prone to respond to cortistatin therapy comprising (i) determining whether the patient has a mutation selected from the group consisting of ER-positive, VHL loss-of-function mutation (VHL-negative), HER2 overexpression , EGFR mutation, MET mutation, biomarkers of neuroblastoma; EWS-FLI1, STAT1-pS727, STAT1, or one or a combination of inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2 and if so, (ii) administering an effective amount of a cortistatin derivative, including, for example, a cortistatin derivative as described herein, or a pharmaceutically acceptable salt, oxide, and/or composition thereof.

The method of Y) X), Z) or AA) comprising determining whether a patient will successfully respond to cortistatin therapy using a kit comprising a multinuclear biomarker or biomarker combination under stringent conditions Probes that anneal to nucleotides or antibodies that bind biomarker proteins.

Z) A method for predicting the response of a patient suffering from a tumor or cancer to treatment with cortistatin comprising the steps of:

a. Obtaining a tumor or cancer sample from a patient;

b. Determining the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of: ER positive, VHL loss-of-function mutation (VHL-negative), HER2 Overexpression, EGFR mutation, MET mutation, biomarkers of neuroblastoma; EWS-FLI1, STAT1-pS727, STAT1, or inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2;

c. Determine whether the expression level or amount assessed in b is outside the range of corresponding normal cells, e.g., higher or lower than the range found in corresponding normal cells, or higher or lower than the patient's clinical A certain amount related to the increase or decrease in benefit; and

d. optionally treating the patient with an effective amount of cortistatin, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition.

AA) A method of selecting patients who will respond to cortistatin therapy comprising:

a. Obtaining a patient's tumor or cancer sample;

b. Detecting the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of: ER positive, VHL loss-of-function mutation (VHL-negative), HER2 Overexpression, EGFR mutation, MET mutation, biomarkers of neuroblastoma; STAT1-pS727, STAT1, EWS-FLI1, or inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2;

c. Comparing the expression determined in step b with the expression of the same gene in a control sample comprising a representative number of cortistatin-sensitive responding patients or predictive animal models and a representative number of patients with no or poor response to cortistatin; and

d. If it is determined that the patient is likely to respond to treatment, administering an effective amount of cortistatin, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

BB) A method from X) to AA) comprising a kit for diagnosing a selected gene comprising primers for the amplification of DNA complementary to a gene-specifically encoded RNA and optionally thermostable DNA polymerisation enzyme.

The method of CC) X) to AA) comprising a kit wherein each primer hybridizes to the RNA encoded by the gene or its complement under standard stringent conditions.

DD) The methods of X) to AA), wherein the tumor or cancer is of hematopoietic lineage.

EE) The method of DD), wherein the hematopoietic lineage tumor or cancer is selected from: acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), childhood B-ALL, acute myeloid leukemia ( AML), chronic lymphoblastic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), childhood B-ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin's Lymphoma, Non-Hodgkin's Lymphoma, Burkitt's Lymphoma, AIDS-Related Lymphoma, Chronic Myeloproliferative Disorders, Primary Central Nervous System Lymphoma, T-Cell Lymphoma, Hairy Cell Leukemia, and Multiple Myeloma (MM), or wherein said cells are precursor cells of a hematopoietic neoplasm or cancer such as myelodysplastic syndrome (MDS).

FF) The methods of V) to AA), wherein the tumor or cancer is a tumor or cancer of non-hematopoietic lineage.

GG) The method of FF), wherein the tumor or cancer is breast cancer, ovarian cancer, endometrial cancer, squamous cell carcinoma, angiosarcoma, colon cancer, gastrointestinal tumor, metastasis-prone solid tumor, clear cell carcinoma, renal cell carcinoma cancer or esophageal cancer.

HH) A method of targeted selection and treatment of a patient with a tumor or cancer that may respond to anti-CDK8/19 therapy comprising (i) determining whether the patient has RUNX1 pathway impairment; if so, (ii) administering an effective amount of CDK8/19 inhibitors include, for example, CDK8/19 inhibitors described herein, or pharmaceutically acceptable salts, oxides, and/or compositions thereof.

II) A method of treating RUNX1-impaired tumors or cancers by administering an effective amount of a CDK8/19 inhibitor in a manner and at a dose that produces sufficient up-regulation of proteins transcribed by RUNX1, so that the cells are more normal, less virulent, induce Tumor or cancer differentiation is caused by maturation, arrest of cell growth or apoptosis.

JJ) The method of HH), II), KK) or LL) comprising using a kit to determine whether a patient will successfully respond to anti-CDK8/19 therapy comprising a combination of a biomarker or a biomarker under stringent conditions Probes that anneal to the polynucleotides of the marker combination or antibodies that bind the biomarker proteins.

KK) A method of predicting the response of a tumor or cancer patient to treatment with a CDK8/19 inhibitor comprising:

a. Obtaining a tumor or cancer sample from a patient;

b. Determining the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPα, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10, E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4, IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK, PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3, ZBTB16, and ZCCHC5;

c. Determine whether the expression level or amount assessed in b is outside the range of corresponding normal cells, e.g., higher or lower than the range found in corresponding normal cells, or higher or lower than the patient's clinical A certain amount related to the increase or decrease in benefit; and

d. optionally treating the patient with an effective amount of a CDK8/19 inhibitor, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

LL) A method of selecting a tumor or cancer patient for treatment with a CDK8/19 inhibitor comprising:

a. Obtaining a patient's tumor or cancer sample;

b. Detecting the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of: ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPα, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10, E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4, IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK, PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3, ZBTB16, and ZCCHC5;

c. Comparing the expression determined in step b to the expression of the same gene in a control sample comprising a representative number of CDK8/19-inhibited patients who responded to the CDK8/19 inhibitor or a predictive animal model and a representative number of patients who did not or poorly responded to the CDK8/19 inhibitor; and

d. If it is determined that the patient is likely to respond to treatment, administering an effective amount of a CDK8/19 inhibitor, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

MM) HH) to LL) methods comprising a kit comprising a set of selected genes for diagnosis of impaired RUNX1 pathway, primers for amplifying DNA complementary to RNA specifically encoded by the gene, and any Thermostable DNA polymerase of choice.

NN) HH) to LL) methods comprising a kit comprising a set of primers encoded for amplification and gene specificity for each gene of the selected genome for which the RUNX1 pathway is diagnosed to be impaired RNA-complementary DNA primers, wherein each primer hybridizes to the gene-encoded RNA or its complement under standard stringent conditions.

OH) HH) to NN), wherein the selected biomarker is one or a combination of GATA1, GATA2, C/EBPa, FLI1, FOG1, ETS1, PU.1 and CBFα.

PP) HH) to NN), wherein the selected biomarkers are BCL2, CCNA1, CD44, C/EBPa, CBFβ, CSF1, CXCL10, CXCR4, ETS1, ETS2, FLI1, FOG1, FCER1A, GATA1, GATA2, One or a combination of GFI1B, HEB, IRF1, IRF8, JAG1, LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1 and TNF.

QQ) HH) to NN), wherein the selected biomarkers are constitutive STAT1-pS727, WT1 mutation, TET2 mutation, IDH1 mutation, IDH2 mutation, MLL rearrangement, C/EBPa mutation, CBFβ rearrangement, PU .1 mutation, GATA1 or 2 mutation, ERG translocation, TLX1 overexpression, and TLX3 activation, or a combination thereof.

RR) HH) to NN) methods comprising the use of at least two biomarkers independently selected from the list in KK), OO) and PP).

SS) HH) to QQ) methods comprising using at least three biomarkers independently selected from the lists in KK), OO) and PP).

TT) HH) to QQ) method comprising using at least four biomarkers independently selected from the list in KK), OO) and PP).

UU) HH) to TT) methods comprising the use of a kit to determine whether a patient will successfully respond to CDK8/19 therapy comprising annealing of a polynucleotide under stringent conditions to a biomarker or biomarker combination probes or antibodies that bind biomarker proteins.

VV) A method of predicting the response of a patient suffering from a tumor or cancer to treatment with a CDK8/19 inhibitor comprising the steps of:

a. Obtaining a tumor or cancer sample from a patient;

b. Determining the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of: ER positive, VHL loss-of-function mutation (VHL-negative), HER2 Overexpression, EGFR mutation, MET mutation, biomarkers of neuroblastoma; EWS-FLI1, STAT1-pS727, STAT1, or inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2;

c. determining whether the expression level or amount assessed in b is above or below the range found in the corresponding normal cells, e.g. above or below a certain amount associated with increased or decreased clinical benefit to the patient; and

d. optionally treating the patient with an effective amount of a CDK8/19 inhibitor, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

WW) A method for selecting a tumor or cancer patient that will respond to treatment with a CDK8/19 inhibitor comprising:

a. Obtaining a patient's tumor or cancer sample;

b. Detecting the expression level or amount of one or more biomarkers in a biological sample from the patient, wherein the biomarkers are selected from the group consisting of: ER positive, VHL loss-of-function mutation (VHL-negative), HER2 Overexpression, EGFR mutation, MET mutation, biomarkers of neuroblastoma; EWS-FLI1, STAT1-pS727, STAT1, or inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2;

c. The expression determined in step b is compared to the expression of the same gene in a control sample comprising a representative number of CDK8/19 inhibitors to determine whether the patient is likely to respond to cortistatin treatment Responding patients or predictive animal models and a representative number of patients who did not or poorly responded to CDK8/19 inhibitors; and

d. If it is determined that the patient is likely to respond to treatment, administering an effective amount of a CDK8/19 inhibitor, or a pharmaceutically acceptable salt or oxide thereof, optionally in a pharmaceutically acceptable composition thereof.

XX) The method of VV to WW) comprising a kit for diagnosing a selected gene comprising primers and optionally a thermostable DNA polymerase for amplifying DNA complementary to RNA specifically encoded by the gene .

YY) The method of VV to WW) comprising a kit comprising a set of primers consisting of, for each gene of the selected genome, the DNA complementary to the RNA specifically encoded by that gene Composition of primers, each of which hybridizes to the RNA encoded by the gene or its complement under standard stringent conditions.

ZZ) The method of VV to YY), wherein the tumor or cancer is a tumor or cancer of the hematopoietic lineage.

The method of AAA)ZZ), wherein the tumor or cancer of the hematopoietic lineage is selected from the group consisting of: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), childhood B-ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related lymphoma, chronic myeloproliferative disease, primary central nervous system lymphoma, T-cell lymphoma, hair cell leukemia, and multiple myeloma (MM), or wherein the cells are hematopoietic neoplasms or cancers such as myeloproliferative Precursor cells of dysmorphic syndrome (MDS).

BBB) The methods of VV) to YY), wherein the tumor or cancer is a tumor or cancer of non-hematopoietic lineage.

The method of CCC) BBB), wherein the tumor or cancer is breast cancer, ovarian cancer, endometrial cancer, squamous cell carcinoma, angiosarcoma, colon cancer, gastrointestinal tumor, metastasis-prone solid tumor, clear cell carcinoma, renal cell carcinoma cancer or esophageal cancer. A) The method to CCC), further comprising treating the patient with a second active agent.

The method of DDD) A) to CCC), further comprising treating the patient with a second active agent, wherein the second active agent is selected from the group consisting of a BET inhibitor, a PI3K inhibitor, a Raf inhibitor, a BTK inhibitor, a Bcl-2 inhibitor, a CDK7 Inhibitors, MEK inhibitors or Syk inhibitors.

EEE) A) to CCC) methods, further comprising treating the patient with a second active agent, wherein the second active agent is selected from nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech/Teva), AMP-244 (Amplimmune /GSK), BMS-936559 (BMS) and MEDI4736 (Roche/Genentech) are PD-1 inhibitors.

FFF) A) to CCC) methods, further comprising treating the patient with at least one other active agent, wherein the second active agent is selected from the group consisting of JQ1, I-BET151 (aka GSK1210151A), I-BET762 (aka GSK525762), OTX-015 (also known as MK-8268, IUPAC 6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine -6-acetamide, 4-(4-chlorophenyl)-N-(4-hydroxyphenyl)-2,3,9-trimethyl-), TEN-010, CPI-203, CPI-0610, BET inhibitor of RVX-208 and LY294002.

The methods of GGG)A) to CCC), further comprising treating the patient with a second active agent, wherein the other active agent is an immunomodulator.

HHH) The method of A) to CCC), wherein the additional active agent is an anti-PD1 antibody.

III) The method of A) to CCC), wherein the additional active agent is an anti-CTLA-4 compound, such as ipilimumab (Yervoy) or tremelimumab.

JJJ) A kit as described in any of the embodiments above.

KKK) A combination dosage form of cortistatin and at least one other active agent for use in combination with a diagnostic agent for patient selection.

The invention is further described in the following sections: Cortistatin (Part I), CDK8/18 Inhibitors (Part II), Patient Selection Based on Sample Biomarker Analysis (Part III), Diagnostics and Kits (Part IV Section), Methods and Pharmaceutical Compositions (Part V), Combinations (Part VI) and Examples (Part VI).

I. Cortistatin

The term "cortistatin" or "cortistatin derivative" or "cortistatin analogue" as used herein refers to an inhibitor of CDK8/19 and has a known naturally occurring cortistatin (cortistatin A , B, C, D, E, F, G, H, I, J, K or L) one of the parent nucleus general ring structure or described in one of the following formulas, or is known in the art Compounds believed to be derivatives of cortistatin, including any references described in the background. Cortistatin may be used in the form of pharmaceutically acceptable salts, including quaternary ammonium salts, N-oxides and/or pharmaceutically acceptable compositions, if desired.

A. Cortistatin analogues

In certain embodiments, cortistatin or an analog thereof is of formula (A-1), (A-1′), (A-1″), (A-2′), (A-2″), (A-3′), (A-3″), (D1′), (D1″), (D2′), (D2″), (E1′), (E1″), (E2′), ( E2"), (G1') or (G1") compounds, or pharmaceutically acceptable salts, quaternary ammonium salts, or N-oxides thereof;

in:

W is -N(R ¹ )(R ² ), -OR ^O , =O or =N(R ¹ );

^R is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, any Optionally substituted heteroaryl, -OR ^A , ^-SRA , -N( ^RA ) ₂ , -C(=O) ^RA , -C(=O)OR ^A , -C(=O)N(R ^A ) ₂ , -S(=O) ₂ R ^A or a nitrogen protecting group;

R is ^hydrogen , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, any Select substituted heteroaryl, -C(=O) ^RA , -C(=O)OR ^A , -C(=O)N( ^RA ) ₂ , -S(=O) ₂ R ^A or nitrogen protection base;

or R and R are ^joined to form optionally substituted heterocyclyl or optionally substituted heteroaryl ^;

R ^O is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, any Select substituted heteroaryl, -C(=O) ^RA , -C(=O)OR ^A , -C(=O)N( ^RA ) ₂ or oxygen protecting group;

R ^N is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, any Optionally substituted heteroaryl, -OR ^A , -C(=O) ^RA , -C(=O)OR ^A , -C(=O)N( ^RA ) ₂ , -S(=O) ₂ R ^A or nitrogen protecting group;

^R is hydrogen or optionally substituted alkyl;

R ⁴ is hydrogen, halogen, optionally substituted alkyl, or -Si( ^RA ) ₃ ;

R ^5A is hydrogen, halogen, optionally substituted alkyl, -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ , - OS(=O) ₂ R ^A , -N ₃ , -N(R ^A ) ₂ , -NR ^A C(=O) ^RA , -NR ^A C(=O)OR ^A , -NR ^A C(=O )N(R ^A ) ₂ , -NR ^A S(=O) ₂ R ^A or -C(R ^A ) ₃ ;

R ^5B is hydrogen, halogen, optionally substituted alkyl, or -OR ^A ;

each Represents a single or double bond, as valence permits, subject to the following conditions:

a. When Expressed as (b) represents a double bond, Denoted as (a2) era form key,

b. When When expressed as (c), it represents a double bond, then one of R ^B1 and R ^B2 does not exist, and one of Y ¹ and Y ² does not exist,

c. When Expressed as a form key in the (c) era, then both R ^B1 and R ^B2 exist, and both Y ¹ and Y ² exist,

d. When Expressed as (a1) represents a double bond, then When expressed as (d2) and (a2), each represents a single bond,

e. Immediately Expressed as (a2) represents a double bond, then When expressed as (a1) and (b), each represents a single bond,

f. When Expressed as (d1) represents a double bond, then Denoted as (d2) era form key;

g. When When expressed as (d2), it represents a double bond, then When expressed as (a1) and (d1), each represents a single bond,

Each of R ^B1 and R ^B2 is independently hydrogen, -L ₁ -R ^B3 or -X ^A R ^A , wherein X ^A is -O-, -S- or -N( ^RA )-; or R ^B1 and ^RB2 is attached to form an oxo group, with the proviso that at least one of ^RB1 and ^RB2 is not hydrogen;

L ₁ is a bond, –CH(CH ₃ )(CH ₂ ) ₂ –, –CH(CH ₃ )-CH=CH–, –C(=O)–, –C(=O)O–, –C( =O)S–, –C(=O)N( ^RL )– or -N( ^RL )-(C(R ^LL ) ₂ ) _p -, where ^RL is hydrogen, optionally substituted alkyl or A nitrogen protecting group, each R ^is independently hydrogen, halogen, or optionally substituted alkyl, and p is 0, 1, or 2;

R ^B3 is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, any A substituted heteroaryl is selected; the proviso is that when L is _a bond, then R ^is not hydrogen;

Each ^RA is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted Aryl, optionally substituted heteroaryl, carbonyl, silyl, an oxygen protecting group when attached to oxygen, a sulfur protecting group when attached to sulfur, or a nitrogen protecting group when attached to nitrogen; optionally When attached to N, two ^RA groups can be joined to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl ring; and optionally when R ^B1 and R ^B2 are each -X ^A R ^A , then two ^RA groups can be joined to form an optionally substituted heterocyclyl ring;

^Y1 and ^Y2 are each hydrogen, or ^Y1 is hydrogen and ^Y2 is -OH, or ^Y1 and ^Y2 are linked to form an oxo (=O) group;

In one embodiment, the present invention includes formulas (A-1), (A-1'), (A-1"), (A-2'), (A-2"), (A-3') , (A-3″), (D1′), (D1″), (D2′), (D2″), (E1′), (E1″), (E2′), (E2″), (G1 ') or (G1") and the additional active compounds described herein, and the use of these compounds with isotopic substitution of at least one desired atom in an amount above (ie enriched in) the natural abundance of the isotope. Isotopes are atoms with the same atomic number but different mass numbers, i.e. the same number of protons but different numbers of neutrons.

Examples of isotopes that may be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ F ³¹ P, ³² P, ³⁵ S, ³⁶ CI, ¹²⁵ I. The invention includes various isotopically labeled compounds as defined herein, for example compounds in which radioactive isotopes such ^as3H , ^{13C and14C} are present. These isotopically labeled compounds can be used in metabolic studies (with ¹⁴ C), reaction kinetics studies (such as ² H or ³ H), detection or imaging techniques such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or stromal tissue distribution assays, or radiation therapy of patients. In particular, ^18F -labeled compounds may be particularly desirable for PET or SPECT studies. Isotopically labeled compounds of the invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the following Schemes or Examples and Preparations by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

By way of general example and not limitation, isotopes of ^hydrogen such as deuterium (2H) and tritium ( ^3H ) may be used anywhere in the structures. Alternatively or additionally, isotopes of carbon such ^{as13C and14C} may be used. A typical isotopic substitution is that hydrogen at one or more positions on the molecule is replaced by deuterium to improve the performance of the drug, such as pharmacodynamics, pharmacokinetics, biodistribution, half-life, stability, AUC, Tmax, Cmax, etc. For example deuterium can bond to carbon during metabolism at the site of bond break (alpha deuterium kinetic isotope effect) or near or in close proximity to the site of bond break (beta deuterium kinetic isotope effect).

Isotopic substitution, such as deuterium substitution, may be partial or total. Partial deuterium substitution means that at least one hydrogen is replaced by deuterium. In certain embodiments, the isotope is 90%, 95%, or 99% or more isotopically enriched at any location of interest. In one embodiment, 90%, 95%, or 99% deuterium enrichment is at the desired location. Unless otherwise stated, any point of enrichment above the natural abundance is sufficient to alter the detectable properties of the drug in humans.

In one embodiment, substitution of a deuterium atom for a hydrogen atom within the R group occurs when at least one variable in the R group is hydrogen (eg, ² H or D) or alkyl (eg, CHD, CD ₂ , CD ₃ ). . For example, when any _R group is or contains eg substituted methyl, ethyl or another alkyl group, the alkyl residue may be _deuterated eg _CD3 , _CH2CD3 or _CD2CD3 . In certain other embodiments, when any of the aforementioned R groups is hydrogen, the ^hydrogen may be isotopically enriched as deuterium (ie,2H).

In some embodiments, ^RB1 is deuterium. In some embodiments, R ^B1 comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, ^RB2 is deuterium. In some embodiments, ^RB2 comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). ^In some embodiments, Y is deuterium. In some embodiments, Y ¹ comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, ^Y2 is deuterium. In some embodiments, Y ² comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ³ is deuterium. In some embodiments, R ³ comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). ^In some embodiments, R4 is deuterium. In some embodiments, R ⁴ comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ^5A is deuterium. In some embodiments, R ^5A comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ^5B is deuterium. In some embodiments, R ^5B comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, ^RN is deuterium. In some embodiments, ^RN comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, W comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ^O is deuterium. In some embodiments, R ^O comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ¹ or R ² is deuterium. In some embodiments, R ¹ or R ² comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, the hydrogens on Ring A (see below) are replaced with deuterium. In some embodiments, the hydrogens on ring B are replaced with deuterium. In some embodiments, the hydrogen on ring C is replaced with deuterium. In some embodiments, the hydrogens on ring D are replaced with deuterium.

cortistatin ring marker

In some embodiments, R5 or another position ^on ring A is deuterated by trapping the enolate with a deuterium source such as _D2O or a deuterated acid. In some embodiments, the position of ring B, C, or D is deuterated by reduction of the double bond (a), (b) or (c ₎ , respectively, with a deuterium source (eg, D2, HD, deuterated borohydride). In some embodiments, the ring D position is deuterated by trapping the enolate (eg, for compounds of formula (XXI)) with a deuterium source (eg, _D2O or a deuterated acid).

Quaternary ammonium salts and N-oxides

As used herein, "quaternary ammonium salt" means a nitrogen atom containing four valence bonds (e.g., substituted by four groups which may be hydrogen and/or non-hydrogen groups), such that the nitrogen atom is positively charged and the charge is equal to ^A counterion (eg Xc as defined herein) balances (neutralizes) the amino group.

As used herein, "N-oxide" refers to a group in which the nitrogen atom contains four valence bonds (e.g., substituted by four groups which may be hydrogen and/or non-hydrogen groups, directly attached to the nitrogen atom is the oxidation group ), making the nitrogen atom positively charged, and wherein the oxidizing group balances (neutralizes) the positively charged amino group of the nitrogen atom.

It should be understood that formula (A-1), (A-1'), (A-1"), (A-2'), (A-2"), (A-3') or (A-3" ) may contain a quaternary ammonium salt and/or N-oxide group at any position where an amino group may be located.

In particular, compounds of formula (A-1') or (A-2") wherein W is -N(R ¹ )(R ² ) may contain a quaternary ammonium salt or N-oxide group at the C ₃ position ( Also known as "quaternary C3 amine salts" and "C3N-oxides"), which contain an amino group -NR ₁ R ₂ attached to ring A.

_In certain embodiments, the amino group at C3 Can be quaternary For example to provide a compound of formula (A-QA') or (A-QA"):

in R ¹ , R ² , R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein; and

in:

Y is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

X ^C is a counterion.

A quaternary C3-amine salt can be formed by reacting a free C3-amine with a group Y× ^C , wherein Y is as defined above (e.g., optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally Substituted carbocyclyl or optionally substituted heterocyclyl), and X ^C is a leaving group as defined herein. The resulting counter ion X ^C can be exchanged for another counter ion X ^C by ion exchange methods such as ion exchange chromatography. Exemplary X ^C counterions include, but are not limited to, halide ions (eg, F ⁻ , Cl ⁻ , Br ⁻ , I ⁻ ), NO ₃ ⁻ , ClO ₄ ⁻ , OH ⁻ , H ₂ PO ₄ ⁻ , HSO ₄ ⁻ , Sulfonate ions (such as methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonate-5-sulfonate, ethyl alkane-1-sulfonic acid-2-sulfonic acid, etc.) and carboxylate ions (for example, acetate, acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.). In certain embodiments, Y is optionally substituted alkyl (eg, methyl). In certain embodiments, X ^C is a halide.

In certain embodiments, the quaternary ammonium salt of formula (A-QA') or (A-QA") is β(A-1-QA') or (A-1-QA") or a( A-2-QA') or (A-2-QA") isomer:

in R ¹ , R ² , R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein.

Or, in some embodiments, the amino group at C3 can be formula N-oxides, for example to provide compounds of formula (A-NO') or (A-NO"):

in R ¹ , R ² , R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein.

In certain embodiments, the N-oxide of formula (A-NO') or (A-NO") is β(A-1-NO') or (A-1-NO") or a (A-2-NO') or (A-2-NO") isomer:

in R ¹ , R ² , R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein.

Compounds of formula (A-1') or (A-1")

As defined generally herein, in certain embodiments cortistatin or a cortistatin analog thereof is a compound of formula (A-1') or (A-1"):

or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide; wherein W is -N(R ¹ )(R ² ), -ORO, =O or =N(R ¹ ).

In certain embodiments, W is -N(R ¹ )(R ² ) to provide compounds of the formula

Or its pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide.

In certain embodiments, the compound of formula (A-1-A') or (A-1-A") has the formula:

Or its pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide.

Compounds of formula (A-1-A') or (A-1-A") include cortistatins (i.e. naturally occurring cortistatins), such as cortistatins A, B, C, D, E, F, G, H, J, K and L, wherein R ^5A and R ^5B are each independently -OR ^A , or wherein represented as (d1) or (d2) At least one of represents a double bond.

For example, in certain embodiments wherein R ^5A and R ^5B are each independently -OR ^A , the cortistatin of formula (A-1-A') or (A-1-A") is selected from the group consisting of:

And its pharmaceutically acceptable salts, quaternary ammonium salts and N-oxides.

where denoted as (d1) or (d2) In certain embodiments where at least one of represents a double bond, the cortistatin of formula (A-1-A') or (A-1-A") is selected from the group consisting of:

And its pharmaceutically acceptable salts, quaternary ammonium salts and N-oxides.

wherein R ^5A and R ^5B are each independently -OR ^A or at least one of (d1) or (d2) In certain embodiments representing a double bond, the cortistatin of formula (A-1-A') or (A-1-A") is selected from the group consisting of:

And its pharmaceutically acceptable salts, quaternary ammonium salts and N-oxides.

As described above, native cortistatin and various cortistatin analogs of formula (A-1-A') or (A-1-A") (where R ^5A and R ^5B are each independently -OR ^A , or at least one of which is denoted as (d1) or (d2) represents a double bond) is described in WO/2010/024930, incorporated herein by reference.

The installation of R ^5A at any α-carbon position of the cyclic ketone can be accomplished through ketone enolate capture reactions during the synthesis of natural cortistatin or cortistatin analogs. Ketones can be trapped as enolates followed by subsequent oxidation or amination of the double bond, or reaction of the double bond with the electrophilic carbon C( ^RA ) ₃ -LG (where LG is the leaving group) to provide substitution The ketone product of wherein R ⁵ is -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ , -OS(=O) ₂ R ^A , -N ₃ , -N(R ^A ) ₂ , -NR ^A C(=O) ^RA , -NR ^A C(=O)OR ^A , -NR ^A C(=O)N(R ^A ) _2. -NR ^A S(=O) ₂ R ^A or -C(R ^A ) ₃ . Exemplary conditions considered for enolate trapping include bases (e.g., lithium diisopropylamide (LDA)) and bases in which P is _a silyl group and LG is a leaving group (e.g., trimethylsilyl Chloride) combination of capture reagents P ₁ -LG.

Exemplary oxidation conditions such as -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ or -OS(=O) ₂ Installation of the ^RA group at the R position involves treatment of the trapped enolate with an oxidizing agent such as m ^- chloroperbenzoic acid (MCPBA), ^MoOOPh , or DMSO to provide a substituted ketone in which R is -OH, followed by optional protection , for example by using ^RA -LG, LG-C(=O) ^RA , LG-C(=O)OR ^A , LG-C(=O)N( ^RA ) ₂ or LG-S(=O) ₂ Compounds of ^RA where LG is a leaving group are treated with compounds where R is -OH to provide compounds where R is ^-OR _A (where RA is ^a non ^- hydrogen group), -OC(=O) ^A compound of RA, -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ or -OS(=O) ₂ ^RA .

Exemplary amination conditions, such as _-N3 , -N( ^RA ) ₂ , ^-NRAC (=O) ^RA , ^-NRAC (=O) ^ORA , ^-NRAC (=O ) N( ^RA ) ₂ or -NR ^A S(=O) ₂ R ^A group installed at the R ⁵ position includes treatment with a compound N ₃ -LG (eg triazide) in which LG is the leaving group Trapped _enolates to afford substituted ketones where R5 is ^-N3 . Substituted ketones wherein R ⁵ is -N ₃ can be treated with a reducing agent such as PPh ₃ to provide compounds wherein R ⁵ is -NH ₂ , followed by optional protection, for example by converting the compound wherein R ⁵ is -NH ₂ Use the formula R ^A -LG, LG-C(=O) ^RA , LG-C(=O)OR ^A , LG-C(=O)N( ^RA ) ₂ or LG-S(=O) ₂ R Treatment of compounds of ^A , wherein LG is a leaving group, to provide wherein R ⁵ is -N( ^RA ) ₂ (wherein at least one R ^A is a non-hydrogen group), -NR ^A C(=O) ^RA , A compound of -NR ^A C(=O)OR ^A , -NR ^A C(=O)N( ^RA ) ₂ or -NR ^A S(=O) ₂ R ^A .

In certain embodiments, for each of (d1) and (d2) Represents a single key. In certain embodiments, R ^5B is hydrogen and is each of (d1) and (d2) Represents a single key. where R ^5B is hydrogen and is each of (d1) and (d2) The synthesis of cortistatin analogs representing single bonds is described in PCT/US2014/072365, which is incorporated herein by reference.

In which W is -N(R ¹ )(R ² ), R ^5B is hydrogen and is (d1) and (d2) In certain embodiments, each representing a single bond, provided is a compound of the formula or a pharmaceutically acceptable salt, quaternary ammonium salt, or N-oxide thereof

In certain embodiments, the compound of formula (A-1-B') or (A-1-B") has the formula:

Or its pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide.

where W is =0, R ^5B is hydrogen and is each of (d1) and (d2) In certain embodiments representing a single bond, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

where W is -OR ^O , R ^5B is hydrogen and is each of (d1) and (d2) In certain embodiments representing a single bond, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

In certain embodiments, the compound of formula (A-1-D') or (A-1-D") has the formula:

Or its pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide.

where W is -OR ^O , R ^5B is hydrogen and is each of (d1) and (d2) In certain embodiments representing a single bond, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

In certain embodiments, the compound of formula (A-1-E') or (A-1-E") has the formula:

Or its pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide.

Groups R ¹ and R ²

As generally defined herein, in the formulas (A-1-A'), (A-1-B'), (A-1-E'), (A-1-A"), (A-1-B In certain embodiments of ") or (A- ¹ -E"), R is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocycle radical, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -OR ^A , -SR ^A , -N( ^RA ) ₂ , -C(=O) ^RA , -C(=O)OR ^A , -C(=O)N( ^RA ) ₂ , -S(=O) ₂ ^RA or a nitrogen protecting group.

Furthermore, in certain embodiments of formulas (A-1-A′), (A-1-A″), (A-1-B′) and (A-1-B″), R is ^hydrogen , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted hetero Aryl, -C(=O) ^RA , -C(=O)ORA, -C(=O)N( ^{RA )} ₂ , -S( ^=O)2RA _or a nitrogen protecting group.

In certain embodiments, at least ^one of R and R is ^hydrogen . ^In certain embodiments, R and R are ^both hydrogen. In certain embodiments, ^one of R and R is ^hydrogen and the other is a non-hydrogen group, such as optionally substituted alkyl. In certain embodiments, R ¹ is hydrogen.

^In certain embodiments, at least ^one of R and R is optionally substituted alkyl, such as optionally substituted C _1-6 alkyl. ^In certain embodiments, R and ^R are each independently optionally substituted alkyl. In certain embodiments, R ¹ is optionally substituted alkyl, such as optionally substituted C _1-6 alkyl. ^In certain embodiments, _R and/or ^R are optionally substituted C _alkyl , optionally substituted C _alkyl , optionally substituted C alkyl, optionally substituted C _alkyl , optionally substituted C ₅ alkyl or optionally substituted C ₆ alkyl. In some embodiments, R ¹ and/or R ² are optionally substituted methyl (C ₁ ), optionally substituted ethyl (C ₂ ), optionally substituted n-propyl (C ₃ ), optionally Substituted isopropyl (C ₃ ), optionally substituted n-butyl (C ₄ ) or optionally substituted tert-butyl (C ₄ ). ^In certain embodiments, R and/or R are alkyl substituted with ^one or more halo substituents (eg, fluoro). In certain embodiments, R ¹ and/or R ² is -CH ₃ or -CF ₃ . In certain embodiments, each instance of R ¹ and R ² is independently -CH ₃ or -CF ₃ . In certain embodiments, R ¹ and/or R ² are substituted by one or more halogen (eg, fluorine), amino (-NH ₂ ), substituted amino, hydroxy (-OH), substituted hydroxy, mercapto (- SH), substituted mercapto, or sulfonyl substituent substituted alkyl. ^In certain embodiments, R and/or R are alkyl substituted with optionally substituted carbocyclyl ⁽ e.g., cyclopropyl) or optionally substituted heterocyclyl (e.g., oxetanyl) rings base.

For example, in certain embodiments, at least ^one of R and R is of the ^formula A group, for example to provide a compound of the formula or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

in R ¹ , R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein; and

in:

p is 1, 2, 3, 4, 5 or 6; and

Z is -CH ₂ X ^Z , -CH(X ^Z ) ₂ , -C(X ^Z ) ₃ , –OR ^Z , –SR ^Z , –N(R ^Z ) ₂ , -S(O) ₂ N(R ^Z ) ₂ ,

wherein each R ^Z is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted Aryl, optionally substituted heteroaryl, -C(=O)R ^Z , -C(=O)OR ^Z , -C(=O)N(R ^Z ) ₂ , when attached to oxygen is oxygen Protecting group, a sulfur protecting group when attached to sulfur, or a nitrogen protecting group when attached to nitrogen, optionally when attached to N two R ^Z groups may be attached to form an optionally substituted heterocyclyl or optionally Substituted heteroaryl rings;

Each instance of X ^Z is independently fluoro, chloro, bromo or iodo; and

w is an integer between 1 and 10, inclusive.

In certain embodiments, R ¹ and R ² are independently of the formula group.

In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, w is 1, 2, 3 or 4. In certain embodiments, R ^Z is hydrogen or optionally substituted alkyl (eg, -CH ₃ ). In certain embodiments, Z is -OR ^Z , eg -OH or -OR ^Z , wherein R ^Z is a non-hydrogen group, eg, wherein R ^Z is optionally substituted alkyl such as -CH ₃ . In certain embodiments, Z is -N(R ^Z ) ₂ , such as -NH ₂ , -NHR ^Z or -N(R ^Z ) ₂ , wherein R ^Z is a non-hydrogen group, such as wherein R ^Z is optionally Substituted alkyl such as _-CH3 . In certain embodiments, Z is -CH ₂ X ^Z , -CH(X ^Z ) ₂ , -C(X ^Z ) ₃ , eg, wherein X ^Z is fluoro. In certain embodiments, Z is -S(O) ₂ N(R ^Z ) ₂ , such as -S(O) ₂ NH ₂ or -S(O) ₂ NHCH ₃ .

_In certain embodiments, R and R are joined to form _an optionally substituted heterocyclyl, such as an optionally substituted 3-6 membered heterocyclyl. In certain embodiments, R and R are joined to form optionally substituted _{3 -} membered heterocyclyl, optionally substituted 4-membered heterocyclyl, optionally substituted 5-membered heterocyclyl, or optionally substituted 6-membered heterocyclyl heterocyclyl. In certain embodiments, R and R are joined to form _an optionally substituted ₃ membered heterocyclyl, ie, optionally substituted aziridinyl. In certain embodiments, R and R are joined to form _an optionally substituted ₄ membered heterocyclyl, such as optionally substituted azetidinyl. In certain embodiments, R and R are joined to form _an optionally substituted ₅ -membered heterocyclyl, such as an optionally substituted pyrrolidinyl or an optionally substituted imidazolidine-2,4-dione. In certain embodiments, R and R are joined to form _an optionally substituted ₆ -membered heterocyclyl, such as optionally substituted piperidinyl, optionally substituted tetrahydropyranyl, optionally substituted dihydropyridine Optionally substituted thianyl, optionally substituted piperazinyl, optionally substituted morpholinyl, optionally substituted dithianyl, optionally substituted dioxanyl, or optionally substituted triazine alkyl.

For example, in certain embodiments, ^R and R are joined to form the ^formula , for example to provide a compound of the formula or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

in R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein; and

in:

G is -O-, -S-, -NH-, -NR ⁷ -, -CH ₂ -, -CH(R ⁷ )- or -C(R ⁷ ) ₂ -;

Each R ^is independently halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted Aryl, optionally substituted heteroaryl, amino, substituted amino, hydroxy, substituted hydroxy, mercapto, substituted mercapto, carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group when attached to a nitrogen atom;

Optionally, wherein two ^R groups are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl ring or oxo (=O ) group; and

n is 0, 1, 2, 3 or 4.

^In certain embodiments, R and R are joined to form the ^formula For example, to provide a compound of the following formula or a pharmaceutically acceptable salt thereof, quaternary ammonium salt or N-oxide:

in R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein; and

in:

G is -O-, -S-, -NH-, -NR ⁷ -, -CH ₂ -, -CH(R ⁷ )- or -C(R ⁷ ) ₂ -

Each R ^is independently halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted Aryl, optionally substituted heteroaryl, amino, substituted amino, hydroxy, substituted hydroxy, mercapto, substituted mercapto, carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group when attached to a nitrogen atom;

Optionally, wherein two ^R groups are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl ring or oxo (=O ) group; and

n is 0, 1, 2, 3 or 4.

^In certain embodiments, R and R are joined to form the ^formula A group, for example to provide a compound of the formula or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

in R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein; and

in:

G is -O-, -S-, -NH-, -NR ⁷ -, -CH ₂ -, -CH(R ⁷ )- or -C(R ⁷ ) ₂ -

Each R ^is independently halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted Aryl, optionally substituted heteroaryl, amino, substituted amino, hydroxy, substituted hydroxy, mercapto, substituted mercapto, carbonyl, sulfonyl, sulfinyl, or a nitrogen protecting group when attached to a nitrogen atom;

Optionally, wherein two ^R groups are joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl ring or oxo (=O ) group; and

n is 0, 1, 2, 3 or 4.

In certain embodiments, n is ⁰ , and the ring system formed by the linkage of R1 and R2 is not substituted by ^{an R7} group as defined herein. In certain embodiments, n is 1, 2, 3 or 4, and the ring system is substituted with 1, 2, 3 or ⁴ R groups as defined herein. In certain embodiments, n is 1. In certain embodiments, n is 2. In certain embodiments, n is 3. In certain embodiments, n is 4.

In certain embodiments wherein n is not 0 (i.e., n is 1, 2, ³ , or ⁴ ) and at least one R is attached to a carbon atom, R is halogen (e.g., fluorine), hydroxy, substituted hydroxy, or a carbonyl group (eg -CO ₂ H). In certain embodiments wherein n is not 0 (i.e., n is 1, 2, ³ , or ⁴ ) and the two R groups are attached to the same carbon atom, each of the two R groups is halogen, such as fluorine . In certain embodiments wherein n is not 0 (i.e., n is 1, 2, ³ , or ⁴ ) and two R groups are attached to the same carbon atom, the two R groups are attached to form an optionally substituted A carbocyclyl ring or an optionally substituted heterocyclyl ring (eg, an optionally substituted oxetanyl ring). In certain embodiments wherein n is not 0 (i.e., n is 1, 2, ³ , or ⁴ ) and the two R groups are attached to different carbon atoms, the two R groups are attached to form an optionally substituted Carbocyclyl ring or optionally substituted heterocyclyl ring.

In certain embodiments, G is -O-. In certain embodiments, G is ^-NR7- , eg, wherein ^R7 is optionally substituted alkyl (eg, _-CH3 ). In certain embodiments, G is -CH( ^R7 )- or -C( ^R7 ) _2- , wherein at least one ^R7 is hydroxyl, substituted hydroxyl, or carbonyl (eg, _-CO2H ).

In some embodiments, the group Yes

In some embodiments, the group Yes

In some embodiments, the group Yes

^In certain embodiments, R1 is -S( ⁼ O ^)2RA _and R2 is optionally substituted alkyl.

Group R ^O

As generally defined herein, R ^O is hydrogen or optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally Substituted aryl, optionally substituted heteroaryl, -C(=O) ^RA , -C(=O) ^ORA , -C(=O)N( ^RA ) ₂ or an oxygen protecting group.

In certain embodiments, R ^O is hydrogen.

In certain embodiments, R ^O is optionally substituted alkyl, such as optionally substituted C _1-6 alkyl, such as optionally substituted C ₁ alkyl, optionally substituted C ₂ alkyl, optionally Substituted _C3 alkyl, optionally substituted _C4 alkyl, optionally substituted _C5 alkyl, or optionally substituted _C6 alkyl. In certain embodiments, R ^O is optionally substituted methyl (C ₁ ), optionally substituted ethyl (C ₂ ), optionally substituted n-propyl (C ₃ ), optionally substituted isopropyl (C ₃ ), optionally substituted n-butyl (C ₄ ) or optionally substituted tert-butyl (C ₄ ). In certain embodiments, R ^O is alkyl substituted with one or more halo substituents (eg, fluoro). In certain embodiments, R ^O is -CH ₃ or -CF ₃ . In certain embodiments, R ^O is substituted with one or more of halogen (eg, fluorine), amino (-NH ₂ ), substituted amino, hydroxyl (-OH), substituted hydroxyl, mercapto (-SH), substituted Alkyl substituted with mercapto or sulfonyl substituents. In certain embodiments, R ^O is alkyl substituted with optionally substituted carbocyclyl (eg, cyclopropyl) or optionally substituted heterocyclyl (eg, oxetanyl).

For example, in certain embodiments, R ^O is of the formula A group: for example to provide a compound of the following formula or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

in R ³ , R ⁴ , R ^5A , R ^B1 and R ^B2 are as defined herein; and

in:

p is 1, 2, 3, 4, 5 or 6; and

Z is -CH ₂ X ^Z , -CH(X ^Z ) ₂ , -C(X ^Z ) ₃ , –OR ^Z , –SR ^Z , –N(R ^Z ) ₂ , -S(O) ₂ N(R ^Z ) ₂ ,

wherein each R ^Z is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted Aryl, optionally substituted heteroaryl, -C(=O)R ^Z , -C(=O)OR ^Z , -C(=O)N(R ^Z ) ₂ , oxygen when attached to oxygen A protecting group, a sulfur protecting group when attached to sulfur, or a nitrogen protecting group when attached to nitrogen, optionally when attached to N two R ^Z groups may be attached to form an optionally substituted heterocyclyl or any Optionally substituted heteroaryl rings;

Each of X and ^Z is independently fluorine, chlorine, bromine or iodine; and

w is an integer between 1 and 10, inclusive.

In certain embodiments, p is 1. In certain embodiments, p is 2. In certain embodiments, p is 3. In certain embodiments, w is 1, 2, 3 or 4. In certain embodiments, R ^Z is hydrogen or optionally substituted alkyl (eg, -CH ₃ ). In certain embodiments, Z is -OR ^Z , eg -OH or -OR ^Z , wherein R ^Z is a non-hydrogen group, eg, wherein R ^Z is optionally substituted alkyl such as -CH ₃ . In certain embodiments, Z is -N(R ^Z ) ₂ , such as -NH ₂ , -NHR ^Z or -N(R ^Z ) ₂ , wherein R ^Z is a non-hydrogen group, such as wherein R ^Z is optionally Substituted alkyl such as _-CH3 . In certain embodiments, Z is -CH ₂ X ^Z , -CH(X ^Z ) ₂ , -C(X ^Z ) ₃ , eg, wherein X ^Z is fluoro. In certain embodiments, Z is -S(O) ₂ N(R ^Z ) ₂ , such as -S(O) ₂ NH ₂ or -S(O) ₂ NHCH ₃ .

In certain embodiments of formula (A-1-D') or (A-1-D"), R ^O is -C(=O) ^RA , -C(=O)OR ^A or -C( =O)N( ^RA ) ₂ . In certain embodiments, ^RA is hydrogen or optionally substituted alkyl (eg, —CH ₃ ). For example, in certain embodiments, R ^O is —C( =O) _CH3 , -C(=O) _OCH3 , -C(=O)N( _CH3 ) ₂ or -C(=O) _NHCH3 .

In certain embodiments, R ^O is an oxygen protecting group.

The groups R ³ , R ⁴ , R ^5A , R ^5B and the formula the key

As generally defined herein, ^R3 is hydrogen or optionally substituted alkyl.

In certain embodiments, ^R3 is hydrogen. In certain embodiments, R ³ is optionally substituted alkyl, such as methyl (—CH ₃ ).

As generally defined herein, R4 is hydrogen, halogen, optionally substituted alkyl, or -Si(RA ₎₃ ^{. In} certain embodiments, R4 is hydrogen. In certain embodiments, R ⁴ is optionally substituted alkyl, such as methyl. In certain embodiments, R ⁴ is -Si( ^RA ) ₃ , eg, wherein each of ^RA is independently optionally substituted alkyl or optionally substituted phenyl.

As generally defined herein, R ^5A is hydrogen, halogen, optionally substituted alkyl, -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( R ^A ) ₂ , -OS(=O) ₂ R ^A , -N ₃ , -N(R ^A ) ₂ , -NR ^A C(=O) ^RA , -NR ^A C(=O)OR ^A , - NR ^A C(=O)N( ^RA ) ₂ , -NR ^A S(=O) ₂ R ^A or -C( ^RA ) ₃ . In certain embodiments, R ^5A is hydrogen. In certain embodiments, R ^5A is a non-hydrogen group. In certain embodiments, R ^5A is halo (eg, bromo, iodo, chloro). In certain embodiments, R ^5A is optionally substituted alkyl (eg, -CH ₃ ). In certain embodiments, R ^5A is -OR ^A (eg -OH, -OCH ₃ ).

In certain embodiments, R ^5A is hydrogen, halo, optionally substituted alkyl, or -OR ^A .

In certain embodiments, R ^5A is -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ , -OS(=O ) ₂ R ^A .

In certain embodiments, R ^5A is -N ₃ , -N( ^RA ) ₂ , -NR ^A C(=O) ^RA , -NR ^A C(=O)OR ^A , -NR ^A C(= O)N( ^RA ) ₂ or -NR ^A S(=O) ₂ R ^A .

In certain embodiments, R ^5A is -C( ^RA ) ₃ .

In certain embodiments, group R ^5A is in the alpha (down) configuration. In certain embodiments, group R ^5A is in the beta (up) configuration.

As generally defined herein, R ^5B is hydrogen, halo, optionally substituted alkyl, or —OR ^A . In certain embodiments, R ^5B is hydrogen. In certain embodiments, R ^5B is a non-hydrogen group. In certain embodiments, R ^5B is halo (eg, bromo, iodo, chloro). In certain embodiments, R ^5B is optionally substituted alkyl, such as methyl. In certain embodiments, R ^5B is -OR ^A , eg -OH. In certain embodiments, R ^5B is not -OR ^A .

In certain embodiments, at least one of ^R5A and ^R5B is hydrogen. In certain embodiments, ^R5A is hydrogen and ^R5B is non-hydrogen. In certain embodiments, ^R5A is non-hydrogen and ^R5B is hydrogen. In certain embodiments, each of ^R5A and ^R5B is hydrogen.

In certain embodiments, at least one of ^R5A and ^R5B is halogen (eg, bromo, iodo, chloro). In certain embodiments, at least one of ^R5A and ^R5B is optionally substituted alkyl, such as methyl.

In certain embodiments, at least one of ^R5A and ^R5B is ^-ORA , eg -OH. In certain embodiments, ^R5A is ^-ORA , eg -OH, and ^R5B is hydrogen. In certain embodiments, ^R5A is hydrogen and ^R5B is ^-ORA , eg -OH. In certain embodiments, each of ^R5A and ^R5B is ^-ORA , eg -OH. In certain embodiments, neither R ^5A nor R ^5B is -OR ^A .

Expressed as each of (a1), (a2), (b), (c), (d1) and (d2) Usually represents a single bond or a double bond, when the price allows, then

when expressed as (c) When representing a double bond, then one of R ^B1 and R ^B2 does not exist, and one of Y ¹ and Y ² does not exist,

when expressed as (c) When representing a single bond, both R ^B1 and R ^B2 exist, and both Y ¹ and Y ² exist,

When expressed as (a1) When representing a double bond, it is expressed as (d2) and (a2) Each represents a single bond,

When expressed as (a2) When representing a double bond, it is expressed as (a1) and (b) Each represents a single bond,

When expressed as (d2) When representing a double bond, it is expressed as (a1) and (d1) Each represents a single bond.

In certain embodiments, the bond represented as (a2) is a single key. In certain embodiments, the bond represented as (a1) is a double bond. In certain embodiments, the bond represented as (b) is a double bond. In certain embodiments, each of (a1) and (b) is a double bond. In certain embodiments, the bond represented as (c) is a single key. In certain embodiments, the bond represented as (d2) is a single key. In certain embodiments, the bond represented as (d1) is a single key.

In certain embodiments, ^R3 is methyl, ^{R4 is hydrogen, R5A is} hydrogen, and the bond denoted as (c) is a single bond.

^In other embodiments, ^R3 is methyl, R4 is hydrogen, the bond denoted as (c) is a double bond, and ^RB2 is absent.

Group R ^B1 and R ^B2

As generally defined herein, each of R ^B1 and R ^B2 is independently hydrogen, -L ₁ -R ^B3 or -X ^A R ^A , wherein X ^A is -O-, -S- or -N(R ^A ) -; or R ^B1 and R ^B2 are joined to form an oxo group, provided that at least one of R ^B1 and R ^B2 is not hydrogen;

L ₁ is a bond, –CH(CH ₃ )(CH ₂ ) ₂ –, –CH(CH ₃ )-CH=CH–, –C(=O)–, –C(=O)O–, –C( =O)S–, –C(=O)N( ^RL )– or -N( ^RL )-(C(R ^LL ) ₂ ) _p -, where ^RL is hydrogen, optionally substituted alkyl or A nitrogen protecting group, each R ^is independently hydrogen, halogen, or optionally substituted alkyl, and p is 0, 1, or 2; and

R ^B3 is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or any A substituted heteroaryl is selected with the proviso that when L is _a bond, then R ^is not hydrogen.

In certain embodiments, at least one of R ^B1 and R ^B2 is -L ₁ -R ^B3 . In certain embodiments, when expressed as (c) When representing a single bond, then R ^B1 is -L ₁ -R ^B3 and R ^B2 is hydrogen or -X ^A R ^A (eg -OR ^A ).

In certain embodiments, L is _a bond, and ^R is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted hetero Cyclic, optionally substituted aryl, or optionally substituted heteroaryl.

In certain embodiments, ^RB3 is a cyclic group, eg, ^RB3 is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, ^RB3 is a non-aromatic cyclic group, eg, in certain embodiments, ^RB3 is optionally substituted carbocyclyl or optionally substituted heterocyclyl. In certain embodiments, ^RB3 is an aromatic cyclic group, eg, in certain embodiments, ^RB3 is optionally substituted aryl or optionally substituted heteroaryl.

In certain embodiments, R ^B3 is optionally substituted aryl, such as optionally substituted C _6-14 aryl. In certain embodiments, ^RB3 is optionally substituted phenyl. In certain embodiments, ^RB3 is optionally substituted naphthyl. In certain embodiments, ^RB3 is optionally substituted phenyl fused to an optionally substituted heterocyclyl ring; such as optionally substituted phenyltetrahydroisoquinolinyl. With respect to optionally substituted aryl ring systems comprising fused heterocyclyl rings, it is understood that the point of attachment to the parent molecule is on the aryl (eg phenyl) ring.

In certain embodiments, ^RB3 is optionally substituted heteroaryl, eg, optionally substituted 5-14 membered heteroaryl. In certain embodiments, ^RB3 is optionally substituted 5-membered heteroaryl or optionally substituted 6-membered heteroaryl. In certain embodiments, ^RB3 is optionally substituted bicyclic heteroaryl, such as optionally substituted 5,6-bicyclic heteroaryl or optionally substituted 6,6-bicyclic heteroaryl. In certain embodiments, ^RB3 is an optionally substituted 5,6-bicyclic heteroaryl or an optionally substituted 6,6-bicyclic heteroaryl ring system selected from optionally substituted naphthyridinyl , optionally substituted pteridinyl, optionally substituted quinolinyl, optionally substituted isoquinolinyl, optionally substituted cinnolinyl, optionally substituted quinoxalinyl, optionally substituted phthalazinyl and optionally substituted quinazolinyl. In certain embodiments, the point of attachment of ^RB3 is through a nitrogen atom.

In certain embodiments, wherein R ^B3 is optionally substituted aryl or optionally substituted heteroaryl, -L ₁ -O ^R3 is selected from the group consisting of:

in:

Each R ^6A is independently halogen, -NO ₂ , -CN, -OR ^6C , -SR ^6C , -N(R ^6C ) ₂ , -C(=O)R ^6C , -C(=O)OR ^6C , -C(=O)N(R ^6C ) ₂ , optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl , optionally substituted aryl or optionally substituted heteroaryl;

each R ^is independently hydrogen, optionally substituted alkyl or a nitrogen protecting group when attached to nitrogen;

wherein each R ^is independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted An aryl, an optionally substituted heteroaryl, an oxygen protecting group when attached to oxygen, a sulfur protecting group when attached to sulfur or a nitrogen protecting group when attached to nitrogen, optionally when attached to N , two ^R groups may be joined to form an optionally substituted heterocyclyl or an optionally substituted heteroaryl ring; and

m is 0 or an integer between 1-4, including 1 and 4.

In certain embodiments, m is 0. In certain embodiments, m is 1, 2, 3 or 4. In certain embodiments, wherein m is 1, 2, 3, or 4, at least one R ^6A is halogen (eg, fluoro), —OR ^6C , —SR ^6C , or —N(R ^6C ) ₂ .

In certain embodiments, as described herein, L is _a bond or -C(=O)N( ^{RL)-, wherein R L is} hydrogen or optionally substituted alkyl (eg, methyl), and R ^B3 is optionally substituted aryl or optionally substituted heteroaryl.

Formula (A-2′), (A-2″), (A-3′), (A-3″), (D1′), (D1″), (D2′), (D2″), ( Compounds of E1'), (E1"), (E2'), (E2"), (G1') and (G1")

As generally defined herein, in certain embodiments, cortistatin or a cortistatin analog thereof is a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

As generally defined herein, ^RN is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally Substituted aryl, optionally substituted heteroaryl, -OR ^A , -C(=O) ^RA , -C(=O)OR ^A , -C(=O)N( ^RA ) ₂ , -S (=O) ₂ R ^A or a nitrogen protecting group.

In certain embodiments, ^RN is optionally substituted alkyl, e.g., optionally substituted C _1-6 alkyl, e.g., optionally substituted C ₁ alkyl, optionally substituted C ₂ alkyl, any _A substituted C3 alkyl, an optionally substituted _C4 alkyl, an optionally substituted _C5 alkyl or an optionally substituted _C6 alkyl is selected. In certain embodiments, R ^O is optionally substituted methyl (C ₁ ), optionally substituted ethyl (C ₂ ), optionally substituted n-propyl (C ₃ ), optionally substituted isopropyl (C ₃ ), optionally substituted n-butyl (C ₄ ), or optionally substituted tert-butyl (C ₄ ).

In certain embodiments, ^RN is -C(=O) ^RA , -C(=O)OR ^A , or -C(=O)N( ^RA ) ₂ . In certain embodiments, ^RA is hydrogen or optionally substituted alkyl (eg, _-CH3 ). For example, in certain embodiments, R ^N is -C(=O) _CH3 , -C(=O) _OCH3 , -C(=O)N( _CH3 ) ₂ , or -C(=O)NHCH ₃ .

In certain embodiments, ^RN is a nitrogen protecting group.

In certain embodiments, ^RN is hydrogen.

In certain embodiments, the compound of formula (A-2') or (A-2") is a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

In certain embodiments, the compound of formula (A-3') or (A-3") is a compound of the formula or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

In certain embodiments, the compound has formula (G1') or (G1"). A compound of formula (G1') or (G1") can be reduced to formula (A-1') as described in the scheme below or the ketone of the compound of (A-1").

For example, a starting ketone can optionally be captured as an enolate (e.g., by treatment with a base and a P1 _- LG group, where P1 is _a silyl group and LG is a leaving group), followed by subsequent bis Oxidation or amination of the bond, or reaction of the double bond with the electrophilic carbon C( ^RA ) ₃ -LG where LG is the leaving group to provide substituted ketone products where R5 is a non ^- hydrogen group such as halogen, -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ , -OS(=O) ₂ R ^A , -N ₃ , - N(R ^A ) ₂ , -NR ^A C(=O) ^RA , -NR ^A C(=O)OR ^A , -NR ^A C(=O)N(R ^A ) ₂ , -NR ^A S(= O) ₂ R ^A or –C(R ^A ) ₃ .

Ketones can be reduced under Wolff-Kishner reducing conditions to provide compounds of formula (G1') and (G1"). Exemplary Wolff-Kishner conditions are described in Furrow, M.E.; Myers, A.G. (2004), "Practical Procedures for the Preparation of N-tert-Butyldimethylsilylhydrazones and Their Use in Modified Wolff-Kishner Reductions and in the Synthesis of Vinyl Halides and gem-Dihalides" Journal of the American Chemical Society 126(17):5436-5445, which is incorporated herein by reference.

Exemplary compound

Various combinations of certain embodiments are further contemplated herein.

For example, where the group -L ₁ -R ^B3 is of formula and wherein L is _a bond, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

in R ^N , R ¹ , R ² , R ³ , R ⁴ , R ^5A , R ^6A and m are as defined herein.

^In certain embodiments wherein R and R are joined to form ^an optionally substituted heterocyclyl, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

wherein R ⁷ , R ^6A , n and m are as defined herein. In certain embodiments, G is O. In certain embodiments, G is N- _CH3 . In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, n is 0. In certain embodiments, n is 1.

^In certain embodiments wherein R and R are joined to form ^an optionally substituted heterocyclyl, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

wherein R ⁷ , R ^6A , n and m are as defined herein. In certain embodiments, G is _-CH2- . In certain embodiments, m is 0. In certain embodiments, m is 1. In certain embodiments, n is 0. In certain embodiments, n is 1.

^In certain embodiments wherein each ^of _R and R is -CH, there is provided a compound of the formula, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof:

wherein R ^6A and m are as defined herein. In certain embodiments, m is 0. In certain embodiments, m is 1.

In certain embodiments wherein ^{one of} R and R is ^hydrogen , and the other of _R and R is -CH, there is provided a compound of the ^formula , or a pharmaceutically acceptable salt thereof, quaternary Amine salt or N-oxide:

wherein R ^6A and m are as defined herein. In certain embodiments, m is 0. In certain embodiments, m is 1.

Exemplary compounds of formula (A-1-B') or (A-1-B") include, but are not limited to:

And its pharmaceutically acceptable salt, quaternary ammonium salt and N-oxide, such as the N-oxide of the following formula:

Exemplary compounds of formula (A-1-C') or (A-1-C") include, but are not limited to:

And its pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide.

Exemplary compounds of formula (A-1-D') or (A-1-D") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula (A-1-E') or (A-1-E") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula (A-2') or (A-2") and (A-3') or (A-3") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula (D1') or (D1") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula (D2') or (D2") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula (E1') or (E1") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

Exemplary compounds of formula (E2') or (E2") include, but are not limited to:

and pharmaceutically acceptable salts thereof.

B. Chemical Definition

Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75th Edition, inside cover), and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry as well as specific functional moieties and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987.

The compounds described herein may contain one or more asymmetric centers and thus exist in various stereoisomeric forms, such as enantiomers and/or diastereomers. Stereoisomers or mixtures thereof with double bonds in the Z or E configuration are also contemplated. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of mixtures of stereoisomers, including racemic mixtures and enriched A mixture of one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and chiral salt formation and crystallization; or preferred isomers can be prepared by asymmetric synthesis . See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E.L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962 ); and Wilen, S.H. Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present invention also includes the compounds as individual isomers substantially free of other isomers, or as a mixture of various isomers.

According to the invention, it is possible to use isomer mixtures containing various isomer ratios. For example, in the case of a mixture of only two isomers, containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2 , 99:1 or 100:0 isomer ratio mixtures are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that for more complex mixtures of isomers, similar ratios can be expected. The mixture may contain two enantiomers, two diastereomers or a mixture of diastereomers and enantiomers.

For example, if a specific enantiomer of a compound described herein is desired, it can be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, wherein the resulting mixture of diastereoisomers is separated and the auxiliary group Cleavage to provide the pure desired enantiomer. In some embodiments, the compounds described herein are prepared by asymmetric synthesis using enzymes. Enantiomers and diastereomers can be separated by fractional crystallization or chromatography (eg, HPLC with a chiral column). Alternatively, where the molecule contains a basic functional group such as amino or an acidic functional group such as carboxyl, diastereoisomeric salts are formed with suitable optically active acids or bases and then analyzed by fractional crystallization or chromatography well known in the art. The diastereomers formed by means of subsequent recovery of the pure enantiomer.

In some embodiments, the carbon to which ^RB1 or ^RB2 is attached is in the (S) configuration. In some embodiments, the carbon to which ^RB1 or ^RB2 is attached is in the (R) configuration. In some embodiments, the carbon to which ^RB1 or ^RB2 is attached has the same configuration as a naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which ^RB1 or ^RB2 is attached has the opposite configuration of naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which Y ¹ or Y ² is attached is in the (S) configuration. In some embodiments, the carbon to which Y ¹ or Y ² is attached is in the (R) configuration. In some embodiments, the carbon to which ^Y1 or ^Y2 is attached has the same configuration as a naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which ^Y1 or ^Y2 is attached has the opposite configuration of naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which ^R is attached is in the (S) configuration. In some embodiments, the carbon to which ^R is attached is in the (R) configuration. In some embodiments, the carbon to which ^R3 is attached has the same configuration as a naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which ^R3 is attached has the opposite configuration of naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which R ^5B is attached is in the (S) configuration. In some embodiments, the carbon to which R ^5B is attached is in the (R) configuration. In some embodiments, the carbon to which R ^5B is attached has the same configuration as a naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which R ^5B is attached has the opposite configuration of naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which R ^5A is attached is in the (S) configuration. In some embodiments, the carbon to which R ^5A is attached is in the (R) configuration. In some embodiments, the carbon to which ^R5A is attached has the same configuration as a naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which ^R5A is attached has the opposite configuration of naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which W is attached is in the (S) configuration. In some embodiments, the carbon to which W is attached is in the (R) configuration. In some embodiments, the carbon to which W is attached has the same configuration as naturally occurring cortistatin (eg, cortistatin A, cortistatin B). In some embodiments, the carbon to which W is attached has the opposite configuration of naturally occurring cortistatin (eg, cortistatin A, cortistatin B).

In some embodiments, the carbon to which ^RB1 is attached is in the (R) configuration. In some embodiments, R ^B1 comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, ^RB2 is deuterium. In some embodiments, ^RB2 comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). ^In some embodiments, Y is deuterium. In some embodiments, Y ¹ comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, ^Y2 is deuterium. In some embodiments, Y ² comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ³ is deuterium. In some embodiments, R ³ comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). ^In some embodiments, R4 is deuterium. In some embodiments, R ⁴ comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ^5A is deuterium. In some embodiments, R ^5A comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ^5B is deuterium. In some embodiments, R ^5B comprises an isotopically enriched atom (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, ^RN is deuterium. In some embodiments, ^RN comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, W comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ^O is deuterium. In some embodiments, R ^O comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, R ¹ or R ² is deuterium. In some embodiments, R ¹ or R ² comprises isotopically enriched atoms (eg, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁸ F). In some embodiments, the hydrogens on Ring A (see below) are replaced with deuterium. In some embodiments, the hydrogens on ring B are replaced with deuterium. In some embodiments, the hydrogen on ring C is replaced with deuterium. In some embodiments, the hydrogens on ring D are replaced with deuterium.

Unless otherwise indicated, structures depicted herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having structures of the present invention other than substituting deuterium or tritium for hydrogen, substituting18F ^for19F , or substituting13C ^{or14C -} enriched ^carbons are within the scope of the present disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

When a range of values is listed, every value and subrange within the range is intended to be encompassed. For example, "C _1-6 alkyl" is intended to cover C ₁ alkyl, C ₂ alkyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, C ₆ alkyl, C _1-6 alkyl, C _1–5 alkyl, C _1–4 alkyl, C _1–3 alkyl, C _1–2 alkyl, C _2–6 alkyl, C _2–5 alkyl, C _2–4 alkyl, C _2-3 alkyl, C _3-6 alkyl, C _3-5 alkyl, C _3-4 alkyl, C _4-6 alkyl, C _4-5 alkyl and C _5-6 alkyl.

As used herein, the term "aliphatic" refers to alkyl, alkenyl, alkynyl and carbocyclyl. Likewise, the term "heteroaliphatic" as used herein refers to heteroalkyl, heteroalkenyl, heteroalkynyl and heterocyclyl.

As used herein, "alkyl" refers to a group of linear or branched saturated hydrocarbon radicals ("C _1-10 alkyl") having 1 to 10 carbon atoms. In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C _1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C _1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C _1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C _1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C _1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C _1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C _1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C _1-2 alkyl”). In some embodiments, an alkyl group has ₁ carbon atom ("C alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C _2-6 alkyl”). Examples of C _1-6 alkyl groups include methyl (C ₁ ), ethyl (C ₂ ), n-propyl (C ₃ ), isopropyl (C ₃ ), n-butyl (C ₄ ), t-butyl (C ₄ ), sec-butyl (C ₄ ), isobutyl (C ₄ ), n-pentyl (C ₅ ), 3-pentyl (C ₅ ), pentyl (C ₅ ), neopentyl (C ₅ ), 3-methyl-2-butyl (C ₅ ), tert-amyl (C ₅ ) and n-hexyl (C ₆ ). Other examples of alkyl groups include n-heptyl (C ₇ ), n-octyl (C ₈ ), and the like. Unless otherwise indicated, each alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents ("substituted alkyl"). In certain embodiments, the alkyl group is unsubstituted C _1-10 alkyl (eg, —CH ₃ ). In certain embodiments, the alkyl group is a substituted C _1-10 alkyl group.

As used herein, "haloalkyl" is a substituted alkyl group as defined herein wherein one or more hydrogen atoms are independently replaced by a halogen such as fluorine, bromine, chlorine or iodine. "Perhaloalkyl" is a subset of haloalkyl and refers to an alkyl group in which all hydrogen atoms are independently replaced by a halogen such as fluorine, bromine, chlorine or iodine. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms (“C _1-8 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms (“C _1-6 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C _1-4 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C _1-3 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C _1-2 haloalkyl”). In some embodiments, all haloalkyl hydrogen atoms are replaced with fluorine to provide a perfluoroalkyl group. In some embodiments, all haloalkyl hydrogen atoms are replaced with chlorine to provide a "perchloroalkyl" group. Examples of haloalkyl include -CF ₃ , -CF ₂ CF ₃ , -CF ₂ CF ₂ CF ₃ , -CCl ₃ , -CFCl ₂ , -CF ₂ Cl and the like.

As used herein, "heteroalkyl" refers to an alkyl group as defined herein, which further includes within the parent chain (i.e. intervening between adjacent carbon atoms) and/or at one or more termini of the parent chain At least one heteroatom selected from oxygen, nitrogen or sulfur (eg, 1, 2, 3 or 4 heteroatoms) is placed at the position. In certain embodiments, heteroalkyl refers to a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC _1-10 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC _1-9 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC _1-8 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC _1-7 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC _1-6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC _1-5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC _1-4 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC _1-3 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC _1-2 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and ₁ heteroatom ("heteroC alkyl"). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC _2-6 alkyl”). Unless otherwise specified, each heteroalkyl group is independently unsubstituted ("unsubstituted heteroalkyl") or substituted with one or more substituents ("substituted heteroalkyl"). In certain embodiments, heteroalkyl is unsubstituted heteroC _1-10 alkyl. In certain embodiments, heteroalkyl is substituted heteroC _1-10 alkyl.

As used herein, "alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon double bonds (eg, 1, 2, 3, or 4 double bonds) . In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C _2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C _2-8 alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C _2-7 alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C _2-6 alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C _2-5 alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C _2-4 alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C _2-3 alkenyl”). In some embodiments, an alkenyl group has ₂ carbon atoms ("C alkenyl"). The one or more carbon-carbon double bonds may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C _2-4 alkenyl include ethenyl (C ₂ ), 1-propenyl (C ₃ ), 2-propenyl (C ₃ ), 1-butenyl (C ₄ ), 2-butenyl ( C ₄ ), butadienyl (C ₄ ), etc. Examples of the C _2-6 alkenyl group include the aforementioned C _2-4 alkenyl group as well as pentenyl (C ₅ ), pentadienyl (C ₅ ), hexenyl (C ₆ ) and the like. Other examples of alkenyl include heptenyl (C ₇ ), octenyl (C ₈ ), octatrienyl (C ₈ ), and the like. Unless otherwise specified, each alkenyl group is independently unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents ("substituted alkenyl"). In certain embodiments, alkenyl is unsubstituted C _2-10 alkenyl. In certain embodiments, alkenyl is a substituted C _2-10 alkenyl.

As used herein, "heteroalkenyl" refers to an alkenyl group as defined herein, which further includes within the parent chain (i.e. intervening between adjacent carbon atoms) and/or at one or more termini of the parent chain Positionally placed at least one heteroatom selected from oxygen, nitrogen or sulfur (eg, 1, 2, 3 or 4 heteroatoms). In certain embodiments, heteroalkenyl refers to a group having 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (" _heteroC2-10 alkenyl") . In some embodiments, a heteroalkenyl has 2 to 9 carbon atoms, at least one double bond, and one or more heteroatoms within the parent chain (" _heteroC alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“ _heteroC alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“ _heteroC alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“ _heteroC alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“ _heteroC alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“ _heteroC alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“ _heteroC alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“ _heteroC alkenyl”). Unless otherwise indicated, each heteroalkenyl group is independently unsubstituted ("unsubstituted heteroalkenyl") or substituted with one or more substituents ("substituted heteroalkenyl"). In certain embodiments, heteroalkenyl is unsubstituted _heteroC2-10 alkenyl. In certain embodiments, heteroalkenyl is substituted _heteroC2-10 alkenyl.

As used herein, "alkynyl" refers to a straight or branched hydrocarbon group having 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (eg, 1, 2, 3, or 4 triple bonds) , ("C _2-10 alkynyl"). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (" _C2-9 alkynyl"). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C _2-8 alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C _2-7 alkynyl"). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C _2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C _2-5 alkynyl"). In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C _2-4 alkynyl"). In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C _2-3 alkynyl"). In some embodiments, an alkynyl has ₂ carbon atoms ("C alkynyl"). The one or more carbon-carbon triple bonds may be internal (eg in 2-butynyl) or terminal (eg in 1-butynyl). Examples of C _2-4 alkynyl include, but are not limited to, ethynyl (C ₂ ), 1-propynyl (C ₃ ), 2-propynyl (C ₃ ), 1-butynyl (C ₄ ), 2 -butynyl (C ₄ ) and the like. Examples of the C _2-6 alkenyl group include the above-mentioned C _2-4 alkynyl group as well as pentynyl (C ₅ ), hexynyl (C ₆ ) and the like. Additional examples of alkynyl include heptynyl (C ₇ ), octynyl (C ₈ ), and the like. Unless otherwise indicated, each alkynyl group is independently unsubstituted ("unsubstituted alkynyl") or substituted ("substituted alkynyl"). In certain embodiments, the alkynyl group is unsubstituted C _2-10 alkynyl. In certain embodiments, the alkynyl group is a substituted C _2-10 alkynyl group.

As used herein, "heteroalkynyl" refers to an alkynyl group, as defined herein, which further comprises one or more At least one heteroatom (for example, 1, 2, 3 or 4 heteroatoms) selected from oxygen, nitrogen or sulfur placed at each terminal position. In certain embodiments, heteroalkynyl refers to a group having 2-10 carbon atoms, at least one triple bond, and one or more heteroatoms within the parent chain (" _heteroC2-10 alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (" _heteroC alkynyl"). In some embodiments, a heteroalkynyl has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“ _heteroC alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“ _heteroC alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“ _heteroC alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“ _heteroC alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“ _heteroC alkynyl”). In some embodiments, a heteroalkynyl has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“ _heteroC alkynyl”). In some embodiments, a heteroalkynyl has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“ _heteroC alkynyl”). Unless otherwise specified, each heteroalkynyl is independently unsubstituted ("unsubstituted heteroalkynyl") or substituted with one or more substituents ("substituted heteroalkynyl"). In certain embodiments, heteroalkynyl is unsubstituted _heteroC2-10 alkynyl. In certain embodiments, the heteroalkynyl is a substituted _heteroC2-10 alkynyl.

As used herein, " _carbocyclyl " or "carbocyclic" refers to non- A group of an aromatic ring hydrocarbon group. In some embodiments, a carbocyclyl has 3 to 10 ring carbon atoms (“C _3-10 carbocyclyl”). In some embodiments, a carbocyclyl has 3 to 9 ring carbon atoms (“C _3-9 carbocyclyl”). In some embodiments, a carbocyclyl has 3 to 8 ring carbon atoms (“C _3-8 carbocyclyl”). In some embodiments, a carbocyclyl has 3 to 7 ring carbon atoms (“C _3-7 carbocyclyl”). In some embodiments, a carbocyclyl has 3 to 6 ring carbon atoms (“C _3-6 carbocyclyl”). In some embodiments, a carbocyclyl has 4 to 6 ring carbon atoms (“C _4-6 carbocyclyl”). In some embodiments, a carbocyclyl has 5 to 6 ring carbon atoms (“C _5-6 carbocyclyl”). In some embodiments, a carbocyclyl has 5 to 10 ring carbon atoms (“C _5-10 carbocyclyl”). Exemplary C _3-6 carbocyclyls include, but are not limited to, cyclopropyl (C ₃ ), cyclopropenyl (C ₃ ), cyclobutyl (C ₄ ), cyclobutenyl (C ₄ ), cyclopentyl (C ₅ ), cyclopentenyl (C ₅ ), cyclohexyl (C ₆ ), cyclohexenyl (C ₆ ), cyclohexadienyl (C ₆ ), etc. Exemplary C _3-8 carbocyclyls include but are not limited to the above C _3-6 carbocyclyls and cycloheptyl (C ₇ ), cycloheptenyl (C ₇ ), cycloheptadienyl (C ₇ ), Cycloheptatrienyl (C ₇ ), cyclooctyl (C ₈ ), cyclooctenyl (C ₈ ), bicyclo [2.2.1] heptyl (C ₇ ), bicyclo [2.2.2] octyl ( C ₈ ) etc. Exemplary C _3-10 carbocyclyls include but are not limited to the above C _3-8 carbocyclyls and cyclononyl (C ₉ ), cyclononenyl (C ₉ ), cyclodecyl (C ₁₀ ), cyclodecyl Alkenyl (C ₁₀ ), octahydro-1H-indenyl (C ₉ ), decalinyl (C ₁₀ ), spiro[4.5]decyl (C ₁₀ ), etc. As indicated by the preceding embodiments, in certain embodiments, a carbocyclyl is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing fused, bridged, or spiro ring systems such as bicyclic systems ( "bicyclic carbocyclyl") or a tricyclic ring system ("tricyclic carbocyclyl")) and may be saturated or may contain one or more carbon-carbon double or triple bonds. "Carbocyclyl" also includes ring systems in which a carbocycle as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocycle, and in which case the number of carbon Continue to indicate the number of carbons in the carbocyclic system. Unless otherwise specified, each carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted with one or more substituents ("substituted carbocyclyl"). In certain embodiments, the carbocyclyl is an unsubstituted C _3-14 carbocyclyl. In certain embodiments, the carbocyclyl is a substituted C _3-14 carbocyclyl.

In some embodiments, a "carbocyclyl" is a monocyclic saturated carbocyclyl having 3 to 10 ring carbon atoms ("C _3-10 cycloalkyl"). In some embodiments, a cycloalkyl has 3 to 9 ring carbon atoms (“C _3-9 cycloalkyl”). In some embodiments, a cycloalkyl has 3 to 8 ring carbon atoms (“C _3-8 cycloalkyl”). In some embodiments, a cycloalkyl has 3 to 6 ring carbon atoms (“C _3-6 cycloalkyl”). In some embodiments, a cycloalkyl has 4 to 6 ring carbon atoms (“C _4-6 cycloalkyl”). In some embodiments, a cycloalkyl has 5 to 6 ring carbon atoms (“C _5-6 cycloalkyl”). In some embodiments, a cycloalkyl has 5 to 10 ring carbon atoms (“C _5-10 cycloalkyl”). Examples of C _5-6 cycloalkyl include cyclopentyl (C ₅ ) and cyclohexyl (C ₆ ). Examples of the C _3-6 cycloalkyl group include the aforementioned C _5-6 cycloalkyl group as well as cyclopropyl (C ₃ ) and cyclobutyl (C ₄ ). Examples of the C _3-8 cycloalkyl group include the above-mentioned C _3-6 cycloalkyl group as well as cycloheptyl (C ₇ ) and cyclooctyl (C ₈ ). Unless otherwise specified, each cycloalkyl group is independently unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl"). In certain embodiments, cycloalkyl is unsubstituted C _3-10 cycloalkyl. In certain embodiments, cycloalkyl is substituted C _3-10 cycloalkyl.

As used herein, "heterocyclyl" or "heterocyclic" refers to a group of 3 to 14 membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur ("3-14 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valence permits. The heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g. fused, bridged or spiro ring systems, such as bicyclic systems ("bicyclic heterocyclyl") or tricyclic systems ("tricyclic heterocyclyl") Cyclic heterocyclyl")), may be saturated or may contain one or more carbon-carbon double or triple bonds. Heterocyclic polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems wherein a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups, wherein the point of attachment is on the carbocyclyl or heterocyclyl ring, or wherein A ring system in which the heterocyclyl ring is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in this case the number of ring members continues to represent the heterocyclyl The number of ring members in the ring system. Unless otherwise indicated, each heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl is a substituted 3-14 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 -10 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 -8-membered heterocyclyl"). In some embodiments, a heterocyclyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 -6-membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, tetrahydrofuryl, dihydrofuryl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-diketone. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepanyl, and thietanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azacanyl, oxocanyl, and thiecanyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuryl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuryl , tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrobenzopyranyl, octahydroisobenzopyranyl, Decalinyl, Decahydro-1,8-naphthyridinyl, Octahydropyrrolo[3,2-b]pyrrole, Indolinyl, Phthalimide, Naphthalimide Amino, chromanyl, benzopyranyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3, 4–b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4Hthieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridyl, 2,3-dihydrofuro [2,3-b]pyridyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridyl, 4,5,6,7-tetrahydrofuro[3,2- c] pyridyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridyl, 1,2,3,4-tetrahydro-1,6-naphthyridyl, etc.

As used herein, "aryl" refers to a monocyclic or polycyclic (eg, bicyclic or tricyclic) 4n+2 having 6-14 ring carbon atoms and zero heteroatoms in an aromatic ring system. A group of an aromatic ring system (eg, having 6, 10 or 14 electrons shared in a ring array) ("C _6-14 aryl"). In some embodiments, an aryl group has ₆ ring carbon atoms ("C aryl", eg, phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C ₁₀ aryl"; eg, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has ₁₄ ring carbon atoms ("C aryl"; eg, anthracenyl). "Aryl" also includes ring systems in which an aryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the radical or point of attachment is on the aryl ring, and in which case , the number of carbon atoms continues to indicate the number of carbon atoms in the aromatic ring system. Unless otherwise specified, each aryl group is independently unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl"). In certain embodiments, the aryl is an unsubstituted C _6-14 aryl. In certain embodiments, the aryl is a substituted C _6-14 aryl.

"Aralkyl" is a subset of "alkyl" and refers to an alkyl group as defined herein substituted with an aryl group as defined herein, wherein the point of attachment is on the alkyl portion.

As used herein, "heteroaryl" means a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) having ring carbon atoms and 1-4 ring heteroatoms provided in an aromatic ring system A group of 4n+2 aromatic ring systems (e.g., with 6, 10 or 14 π-electrons shared in a ring array), wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-14 metaheteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon atom or a nitrogen atom, as valence permits. Heteroaryl polycyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in which case, The number of ring members continues to refer to the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in which case the ring member The number of represents the number of ring members in a fused polycyclic (aryl/heteroaryl) ring system. A polycyclic heteroaryl group in which one ring does not contain a heteroatom (such as indolyl, quinolinyl, carbazolyl, etc.) can be attached to any ring, that is, a ring with a heteroatom (such as 2-indolyl Indolyl) or rings containing no heteroatoms (eg 5-indolyl).

In some embodiments, heteroaryl is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen , oxygen and sulfur ("5-10 membered heteroaryl"). In some embodiments, heteroaryl is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen , oxygen and sulfur ("5-8 membered heteroaryl"). In some embodiments, heteroaryl is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen , oxygen and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise indicated, each heteroaryl is independently unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl"). In certain embodiments, the heteroaryl is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyrrolyl, furyl, and thienyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzothienyl, Furyl, Benzisofuryl, Benzimidazolyl, Benzoxazolyl, Benzisoxazolyl, Benzoxadiazolyl, Benzothiazolyl, Benzisothiazolyl, Benzothiadiazole group, indolizinyl group and purinyl group. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazole Linyl. Exemplary tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

"Heteroaralkyl" is a subset of "alkyl" and refers to an alkyl group as defined herein substituted with a heteroaryl group as defined herein, wherein the point of attachment is on the alkyl portion.

As used herein, the term "partially unsaturated" refers to ring moieties that contain at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (eg, aryl or heteroaryl moieties) as defined herein.

As used herein, the term "saturated" refers to a portion of a ring that contains no double or triple bonds, ie the ring contains all single bonds.

Attaching the suffix "-ene" to a group indicates that the group is a divalent moiety, for example, an alkylene is the divalent part of an alkyl, an alkenylene is the divalent part of an alkenyl, and an alkynylene is the divalent part of an alkynyl. A divalent moiety, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, and carbocyclylene is the divalent moiety of heteroalkynyl A divalent part of a group, a heterocyclylene is a divalent part of a heterocyclyl, an arylene is a divalent part of an aryl, and a heteroarylene is a divalent part of a heteroaryl.

It will be understood from the above that alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl as defined herein are optionally in certain embodiments was replaced. Optionally substituted means that it may be a substituted or unsubstituted group (e.g. "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "Unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl, "Substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl or "substituted" or "unsubstituted" "heteroaryl). Generally, the term "substituted" means that at least one hydrogen present on a group is replaced by a permissive substituent, e.g., the substituted substituent results in a stable compound, e.g., does not spontaneously undergo transformations such as by rearrangement, cyclization, elimination or other reactive compounds. Unless otherwise stated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, at each position The substituents are the same or different. The present invention contemplates any and all such combinations to achieve stable compounds. For the purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and/or any suitable substituent as described herein which satisfies the valence of the heteroatom and results in the formation of a stable moiety.

Exemplary substituents include, but are not limited to, halogen, —CN, —NO ₂ , —N ₃ , —SO ₂ H, —SO ₃ H, —OH, —OR ^aa , —ON(R ^bb ) ₂ , —N( R ^bb ) ₂ , –N(R ^bb ) ₃ ⁺ X ^– , –N(OR ^cc )R ^bb , –SH, –SR ^aa , –SSR ^cc , –C(=O)R ^aa , –CO ₂ H, –CHO, –C(OR ^cc ) ₂ , –CO ₂ R ^aa , –OC(=O)R ^aa , –OCO ₂ R ^aa , –C(=O)N(R ^bb ) ₂ , –OC(=O )N(R ^bb ) ₂ , –NR ^bb C(=O)R ^aa , –NR ^bb CO ₂ R ^aa , –NR ^bb C(=O)N(R ^bb ) ₂ , –C(=NR ^bb )R ^aa , –C(=NR ^bb )OR ^aa , –OC(=NR ^bb )R ^aa , –OC(=NR ^bb )OR ^aa , –C(=NR ^bb )N(R ^bb ) ₂ , –OC(= NR ^bb )N(R ^bb ) ₂ , –NR ^bb C(=NR ^bb )N(R ^bb ) ₂ , –C(=O)NR ^bb SO ₂ R ^aa , –NR ^bb SO ₂ R ^aa , –SO ₂ N(R ^bb ) ₂ , –SO ₂ R ^aa , –SO ₂ OR ^aa , –OSO ₂ R ^aa , –S(=O)R ^aa , –OS(=O)R ^aa , –Si(R ^aa ) ₃ , –OSi(R ^aa ) ₃ –C(=S)N(R ^bb ) ₂ , –C(=O)SR ^aa , –C(=S)SR ^aa , –SC(=S)SR ^aa , –SC (=O)SR ^aa , –OC(=O)SR ^aa , –SC(=O)OR ^aa , –SC(=O)R ^aa , –P(=O)(R ^aa ) ₂ , –OP(= O)(R ^aa ) ₂ , –OP(=O)(OR ^cc ) ₂ , –NR ^bb P(=O)(OR ^cc ) ₂ , –P(R ^cc ) ₂ , –OP(R ^cc ) ₂ , –B(R ^aa ) ₂ , –B(OR ^cc ) ₂ , –BR ^aa (OR ^cc ), C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 Alkynyl, hetero C _1-10 alkyl, hetero C _2-10 alkenyl, hetero C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 ^R groups;

Or two geminal hydrogens on a carbon atom are replaced by the following groups: =O, =S, =NN(R ^bb ) ₂ , =NNR ^bb C(=O)R ^aa , =NNR ^bb C(=O)OR ^aa , =NNR ^bb S(=O) ₂ R ^aa , =NR ^bb or =NOR ^cc ;

Each R ^aa is independently selected from C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, hetero C _1-10 alkyl, hetero C _2-10 Alkenyl, hetero C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, or two R ^aa groups connected Form a 3-14 membered heterocyclyl or a 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, Aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 ^R groups;

Each R ^bb is independently selected from hydrogen, —OH, —OR ^aa , —N(R ^cc ) ₂ , —CN, —C(=O)R ^aa , —C(=O)N(R ^cc ) ₂ , –CO ₂ R ^aa , –SO ₂ R ^aa , –C(=NR ^cc )OR ^aa , –C(=NR ^cc )N(R ^cc ) ₂ , –SO ₂ N(R ^cc ) ₂ , –SO ₂ R ^cc , –SO ₂ OR ^cc , –SOR ^aa , –C(=S)N(R ^cc ) ₂ , –C(=O)SR ^cc , –C(=S)SR ^cc –P(=O)(R ^aa ) ₂ , C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, hetero C _1-10 alkyl, hetero C _2-10 alkenyl, hetero C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, or two R ^bb groups linked to form 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and hetero ^Aryl groups are independently substituted by 0, 1, 2, 3, 4 or 5 R groups;

Each R ^cc is independently selected from hydrogen, C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, heteroC _1-10 alkyl, heteroC _{2 -10} alkenyl, hetero C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl, or two R ^cc groups Groups are connected to form a 3-14 membered heterocyclic group or a 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocycle radical, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 ^R groups;

Each R ^dd is independently selected from halogen, -CN, -NO ₂ , -N ₃ , -SO ₂ H, -SO ₃ H, -OH, -OR ^ee , -ON(R ^ff ) ₂ , -N(R ^ff ) ₂ , –N(R ^ff ) ₃ ⁺ X ^– , –N(OR ^ee )R ^ff , –SH, –SR ^ee , –SSR ^ee , –C(=O)R ^ee , –CO ₂ H, – CO ₂ R ^ee , –OC(=O)R ^ee , –OCO ₂ R ^ee , –C(=O)N(R ^ff ) ₂ , –OC(=O)N(R ^ff ) ₂ , –NR ^ff C (=O)R ^ee , –NR ^ff CO ₂ R ^ee , –NR ^ff C(=O)N(R ^ff ) ₂ , –C(=NR ^ff )OR ^ee , –OC(=NR ^ff )R ^ee , –OC(=NR ^ff )OR ^ee , –C(=NR ^ff )N(R ^ff ) ₂ , –OC(=NR ^ff )N(R ^ff ) ₂ , –NR ^ff C(=NR ^ff )N(R ^ff ) ₂ ,–NR ^ff SO ₂ R ^ee ,–SO ₂ N(R ^ff ) ₂ ,–SO ₂ R ^ee ,–SO ₂ OR ^ee ,–OSO ₂ R ^ee ,–S(=O)R ^ee ,– Si(R ^ee ) ₃ , –OSi(R ^ee ) ₃ , –C(=S)N(R ^ff ) ₂ , –C(=O)SR ^ee , –C(=S)SR ^ee , –SC(= S)SR ^ee , –P(=O)(R ^ee ) ₂ , –OP(=O)(R ^ee ) ₂ , –OP(=O)(OR ^ee ) ₂ , C _1-6 alkyl, C _{1 -6} perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, hetero C _1-6 alkyl, hetero C _2-6 alkenyl, hetero C _2-6 alkynyl, C _3-10 carbocycle Base, 3-10 membered heterocyclyl, C _6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbon Cyclic, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 ^Rdd groups; or two geminal ^Rdd substituents can be linked to form =O or = S;

Each R ^ee is independently selected from C _1-6 alkyl, C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, heteroC _1-6 alkyl, heteroC _2-6 Alkenyl, hetero C _2-6 alkynyl, C _3-10 carbocyclyl, C _6-10 aryl, 3-10 membered heterocyclic group and 3-10 membered heteroaryl, wherein each alkyl, alkenyl , alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted with 0, 1, 2, 3, 4, or 5 R ^gg groups ;

Each R ^ff is independently selected from hydrogen, C _1-6 alkyl, C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, heteroC _1-6 alkyl, heteroC _{2 -6} alkenyl, hetero C _2-6 alkynyl, C _3-10 carbocyclyl, 3-10 member heterocyclyl, C _6-10 aryl and 5-10 member heteroaryl, or two R ^ff groups Groups are connected to form a 3-14 membered heterocyclic group or a 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocycle radical, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R ^gg groups; and

Each R ^gg is independently halogen, -CN, -NO ₂ , -N ₃ , -SO ₂ H, -SO ₃ H, -OH, -OC _1-6 alkyl, -ON(C _1-6 alkyl ) ₂ , –N(C _1–6 alkyl) ₂ , –N(C _1–6 alkyl) ₃ ⁺ X ^– , –NH(C _1–6 alkyl) ₂ ⁺ X ^– , –NH ₂ (C _1–6 alkyl) ⁺ X ^– , –NH ₃ ⁺ X ^– , –N(OC _1–6 alkyl)(C _1–6 alkyl), –N(OH)(C _1–6 alkyl), –NH(OH), –SH, –SC _1–6 alkyl, –SS(C _1–6 alkyl), –C(=O)(C _1–6 alkyl), –CO ₂ H, –CO ₂ (C _1–6 alkyl), –OC(=O)(C _1–6 alkyl), –OCO ₂ (C _1–6 alkyl), –C(=O)NH ₂ , –C(= O)N(C _1–6 alkyl) ₂ , –OC(=O)NH(C _1–6 alkyl), –NHC(=O)(C _1–6 alkyl), –N(C _{1– 6} alkyl)C(=O)(C _1–6 alkyl), –NHCO ₂ (C _1–6 alkyl), –NHC(=O)N(C _1–6 alkyl) ₂ , –NHC( =O)NH(C _1–6 alkyl), –NHC(=O)NH ₂ , –C(=NH)O(C _1–6 alkyl), –OC(=NH)(C _1–6 alkyl radical), –OC(=NH)OC _1–6 alkyl, –C(=NH)N(C _1–6 alkyl) ₂ , –C(=NH)NH(C 1–6 alkyl), –C(=NH)NH(C _1–6 alkyl), – C(=NH)NH ₂ , –OC(=NH)N(C _1–6 alkyl) ₂ , –OC(NH)NH(C _1–6 alkyl), –OC(NH)NH ₂ , –NHC (NH)N(C _1–6 alkyl) ₂ , –NHC(=NH)NH ₂ , –NHSO ₂ (C _1–6 alkyl), –SO ₂ N(C _1–6 alkyl) ₂ , – SO ₂ NH (C _1–6 alkyl), –SO ₂ NH ₂ , –SO ₂ C _1–6 alkyl, –SO ₂ OC _1–6 alkyl, –OSO ₂ C _1–6 alkyl, –SOC _1–6 alkyl, –Si(C _1–6 alkyl) ₃ , –OSi(C _1–6 alkyl) ₃ –C(=S)N(C _1–6 alkyl) ₂ , C(=S )NH(C _1–6 alkyl), C(=S)NH ₂ , –C(=O)S(C _1–6 alkyl), –C(=S)SC _1–6 alkyl, –SC (=S)SC _1–6 alkyl, –P(=O)(C _1–6 alkyl) ₂ , –OP(=O)(C _1–6 alkyl) ₂ , –OP(=O)( OC _1–6 alkyl) ₂ , C _1-6 alkane C _1-6 perhaloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, hetero C _1-6 alkyl, hetero C _2-6 alkenyl, hetero C _2-6 alkynyl, C _{3 -10} carbocyclic group, C _6-10 aryl group, 3-10 membered heterocyclic group, 5-10 membered heteroaryl group; or two geminal R ^gg substituents can be connected to form =O or ⁼ S; wherein X- is a counter ion.

In certain embodiments, exemplary substituents are selected from halogen, —CN, —NO ₂ , —N ₃ , —SO ₂ H, —SO ₃ H, —OH, —OR ^aa , —N(R ^bb ) ₂ , –SH, –SR ^aa , –SSR ^cc , –C(=O)R ^aa , –CO ₂ H, –CHO, –CO ₂ R ^aa , –OC(=O)R ^aa , –OCO ₂ R ^aa , –C(=O)N(R ^bb ) ₂ , –OC(=O)N(R ^bb ) ₂ , –NR ^bb C(=O)R ^aa , –NR ^bb CO ₂ R ^aa , –NR ^bb C (=O)N(R ^bb ) ₂ , –C(=O)NR ^bb SO ₂ R ^aa , –NR ^bb SO ₂ R ^aa , –SO ₂ N(R ^bb ) ₂ , –SO ₂ R ^aa , –S (=O)R ^aa , C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, hetero C _1-10 alkyl, hetero C _2-10 alkenyl group, hetero C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclic group, C _6-14 aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, Alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted with 0, 1, 2, 3, 4 or 5 ^Rdd groups.

As used herein, the term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) or iodine (iodo, -I) .

As used herein, a "counterion" is a negatively charged group bonded to a positively charged quaternary amine to maintain electronic neutrality. Exemplary counterions include halides (e.g., F ⁻ , Cl ⁻ , Br ⁻ , I ⁻ ), NO ₃ ⁻ , ClO ₄ ⁻ , OH ⁻ , H ₂ PO ₄ ⁻ , HSO ₄ ⁻ , sulfonate ions (e.g., formazan Sulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonate-5-sulfonate, ethane-1-sulfonate -2-sulfonate, etc.) and carboxylate ions (such as acetate, acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, etc.).

As used herein, "leaving group" is an art-understood term that refers to a molecular fragment that leaves with a pair of electrons in the cleavage of a heterogeneous bond, where the molecular fragment is an anion or a neutral molecule. See eg Smith, March Advanced Organic Chemistry 6th Edition (501-502). Exemplary leaving groups include, but are not limited to, halogen (eg, chlorine, bromine, iodine) and -OSO ₂ R ^aa , wherein R ^aa is as defined herein. The group -OSO ₂ R ^aa includes leaving groups such as tosyl, mesyl and benzenesulfonyl, where R ^aa is optionally substituted alkyl (eg, -CH ₃ ) or optionally substituted aryl ( such as phenyl, tolyl).

As used herein, the term "hydroxyl" or "hydroxyl" refers to the group -OH. Relatedly, the term "substituted hydroxy" or "substituted hydroxy" refers to a hydroxy group in which the oxygen atom directly attached to the parent molecule is replaced by a group other than hydrogen, such groups selected from the group consisting of -OR ^aa , –ON(R ^bb ) ₂ , –OC(=O)SR ^aa , –OC(=O)R ^aa , –OCO ₂ R ^aa , –OC(=O)N(R ^bb ) ₂ , –OC(=NR ^bb )R ^aa , –OC(=NR ^bb )OR ^aa , –OC(=NR ^bb )N(R ^bb ) ₂ , –OS(=O)R ^aa , –OSO ₂ R ^aa , –OSi(R ^aa ) 3. A group of -OP(R ^cc ) ₂ , -OP(=O)(R ^aa ) ₂ and -OP(=O)(OR ^cc ) ₂ , wherein R ^aa , R ^bb and R ^cc are as defined herein .

As used herein, the term "mercapto" or "thio" refers to the group -SH. Relatedly, the term "substituted mercapto" or "substituted thio" refers to a thiol group in which the sulfur atom directly attached to the parent molecule is replaced by a group other than hydrogen, such groups include groups selected from -SR ^aa , -S=SR ^cc , -SC(=S)SR ^aa , -SC(=O)SR ^aa , -SC(=O)OR ^aa and -SC(=O)R ^aa , where R ^aa and R ^cc are as defined herein.

As used herein, the term "amino" refers to the group _-NH2 . Relatedly, the term "substituted amino" refers to a monosubstituted or disubstituted amino group as defined herein. In certain embodiments, a "substituted amino" is a monosubstituted amino or a disubstituted amino.

As used herein, the term "monosubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by one hydrogen and one group other than hydrogen, such groups include -NH(R ^bb ) , –NHC(=O)R ^aa , –NHCO ₂ R ^aa , –NHC(=O)N(R ^bb ) ₂ , –NHC(=NR ^bb )N(R ^bb ) ₂ , –NHSO ₂ R ^aa and – A group of NHP(=O)(OR ^cc ) ₂ wherein R ^aa , R ^bb and R ^cc are as defined herein, and wherein R ^bb of the group -NH(R ^bb ) is not hydrogen.

As used herein, the term "disubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by two groups other than hydrogen, such groups include -N(R ^bb ) ₂ , –NR ^bb C(=O)R ^aa , –NR ^bb CO ₂ R ^aa , –NR ^bb C(=O)N(R ^bb ) ₂ , –NR ^bb C(=NR ^bb )N(R ^bb ) ₂ , A group of -NR ^bb SO ₂ R ^aa and -NR ^bb P(=O)(OR ^cc ) ₂ , wherein R ^aa , R ^bb and R ^cc are as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not replaced by hydrogen.

As used herein, the term "sulfonyl" refers to a group selected from -SO ₂ N(R ^bb ) ₂ , -SO ₂ R ^aa and -SO ₂ OR ^aa , wherein R _aa and R _bb are as defined herein.

As used herein, the term "sulfinyl" refers to the group -S(=O)R ^aa , wherein R ^aa is as defined herein.

As used herein, the term "carbonyl" refers to a group in which the carbon directly attached to the parent molecule is ^sp2 hybridized and replaced by an oxygen, nitrogen or sulfur atom, such groups are selected from, for example, ketones (-C(= O)R ^aa ), carboxylic acid (-CO ₂ H), aldehyde (-CHO), ester (–CO ₂ R ^aa , –C(=O)SR ^aa , –C(=S)SR ^aa ), amide ( –C(=O)N(R ^bb ) ₂ , –C(=O)NR ^bb SO ₂ R ^aa , –C(=S)N(R ^bb ) ₂ ) and imines (–C(=NR ^bb ) R ^aa , -C(=NR ^bb ) OR ^aa ), -C(=NR ^bb )N(R ^bb ) ₂ ), wherein R ^aa and R ^bb are as defined herein.

As used herein, the term "silyl" refers to the group -Si(R ^aa ) ₃ , wherein R ^aa is as defined herein.

As used herein, the term "oxo" refers to the group =O, and the term "thiooxo" refers to the group =S.

Nitrogen atoms may be substituted or unsubstituted as valence permits and include primary, secondary, tertiary and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR ^aa , -N(R ^cc ) ₂ , -CN, -C(=O)R ^aa , -C(=O)N(R ^cc ) ₂ , –CO ₂ R ^aa , –SO ₂ R ^aa , –C(=NR ^bb )R ^aa , –C(=NR ^cc )OR ^aa , –C(=NR ^cc )N(R ^cc ) ₂ , – SO ₂ N(R ^cc ) ₂ , –SO ₂ R ^cc , –SO ₂ OR ^cc , –SOR ^aa , –C(=S)N(R ^cc ) ₂ , –C(=O)SR ^cc , –C( =S)SR ^cc , -P(=O)(R ^aa ) ₂ , C _1-10 alkyl, C _1-10 perhaloalkyl, C _2-10 alkenyl, C _2-10 alkynyl, hetero C _{1 -10} alkyl, hetero C _2-10 alkenyl, hetero C _2-10 alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclyl, C _6-14 aryl and 5-14 membered heteroaryl group, or two R ^cc groups connected to the N atom are connected to form a 3-14 membered heterocyclic group or a 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkene radical, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently substituted by 0, 1, 2, 3, 4 or 5 R ^dd groups, and wherein R ^aa , R ^bb , R ^cc and ^Rdd are as defined above.

In certain embodiments, substituents present on nitrogen atoms are nitrogen protecting groups (also referred to herein as "amino protecting groups"). Nitrogen protecting groups include but are not limited to -OH, -OR ^aa , -N(R ^cc ) ₂ , -C(=O)R ^aa , -C(=O)N(R ^cc ) ₂ , -CO ₂ R ^aa , –SO ₂ R ^aa , –C(=NR ^cc )R ^aa , –C(=NR ^cc )OR ^aa , –C(=NR ^cc )N(R ^cc ) ₂ , –SO ₂ N(R ^cc ) ₂ , –SO ₂ R ^cc , –SO ₂ OR ^cc , –SOR ^aa , –C(=S)N(R ^cc ) ₂ , –C(=O)SR ^cc , –C(=S)SR ^cc , C _{1- 10} Alkyl (eg aralkyl, heteroaralkyl), C _2-10 alkenyl, C _2-10 alkynyl, hetero C _1-10 alkyl, hetero C _2-10 alkenyl, hetero C _2-10 Alkynyl, C _3-10 carbocyclyl, 3-14 membered heterocyclic group, C _6-14 aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, Heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently substituted with ^{0, 1, 2, 3, 4, or 5 R groups, and wherein R aa ,} R ^bb , R ^cc and R ^dd are as defined herein. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd Ed., John Wiley & Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amido groups (eg -C(=O)R ^aa ) include but are not limited to formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3- Phenylpropanamide, Pyridineamide, 3-Pyridylformamide, N-Benzoylphenylalanyl Derivatives, Benzamide, p-Phenylbenzamide, o-Nitrophenylacetamide, o-Nitrophenoxy Acetamide, acetoacetamide, (N'-dithiobenzyloxyamido)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2- Methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutanamide, 3-methyl-3-nitro Nylbutanamide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (eg -C(=O)OR ^aa ) include but are not limited to methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), amino 9-(2-sulfo)fluorenylmethyl formate, 9-(2,7-dibromo)fluorenylmethylcarbamate, 2,7-di-tert-butyl-[9-(10,10- Dioxo-10,10,10,10-tetrahydrothioxanthyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2 ,2,2-Trichloroethyl carbamate (Troc), 2-trimethylsilyl ethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2 ,2-Dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl- 1-(4-biphenyl)ethyl carbamate (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2–(2’– and 4’–pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, tert-butyl carbamate ( BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamon Carbamate (Coc), 4-Nitrocinnamylcarbamate (Noc), 8-Quinolinylcarbamate, N-Hydroxypiperidinylcarbamate, Alkyldithiocarbamate Ester, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl amino Formate, 2,4-Dichlorobenzylcarbamate, 4-Methylsulfinylbenzylcarbamate (Msz), 9-Anthracenylmethylcarbamate, Diphenylmethylcarbamate Carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl) ethyl carbamate, [2-( 1,3-Dithiolane)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate Ester (Bmpc), 2-phosphonoethyl carbamate (Peoc), 2-triphenylphosphonoisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate Base carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl) benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(Trifluoromethyl)-6-chromanylmethylcarbamate (Tcroc) , m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate Base carbamate, phenyl (o-nitrophenyl) methyl carbamate, tert-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, amino Cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate 1,1-dimethyl-3-(N,N-dimethylformylamino)propylcarbamate ester, 1,1-dimethylpropynyl carbamate, bis(2-pyridyl) methyl carbamate, 2-furyl methyl carbamate, 2-iodoethyl carbamate ester, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutane Carbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl ) ethyl carbamate, 1-methyl-1-(p-phenylazophenyl) ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1- Methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2-4,6-tri-tert-butylphenylamino Formate, 4-(trimethylammonium)benzylcarbamate and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g. -S(=O)2R ^aa ) include but are not limited to p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxy Benzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6 -Tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-di Methoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-Trimethylsilylethanesulfonamide (SES), 9-Anthracenesulfonamide, 4-(4',8'-Dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), Benzylsulfonamide , trifluoromethanesulfonamide and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivatives, N'-p-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoyl Phenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-azolin-2-one, N-phthalimide, N-dithiosuccinate Imide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilyl Azacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-di Benzyl-1,3,5-triazepan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[ 2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3- Pyrrolin-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-bis(4-methoxyphenyl)methylamine, N-5-dibenzocycloheptylamine, N-triphenylmethylamine Amine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro- 9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-methylpyridylamino N'-oxide, N-1,1-dimethylthiomethylene Baseamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)tricresyl]methyleneamine, N-( N', N'-dimethylaminomethylene)amine, N,N'-isopropylenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chloro Salicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneimine, N-(5,5-dimethyl-3-oxo- 1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboronic acid derivatives, N-[phenyl(pentaylchromium or tungsten)yl]amine, N-copper chelate, N- Zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphonamide (Dpp), dimethylthiophosphonamide (Mpt), diphenylphosphinethio Amide (Ppt), dialkylphosphoramidate, dibenzylphosphoramidate, diphenylphosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzene Sulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, substituents present on an oxygen atom are oxygen protecting groups (also referred to herein as "hydroxyl protecting groups"). Oxygen protecting groups include but are not limited to –R ^aa , –N(R ^bb ) ₂ , –C(=O)SR ^aa , –C(=O)R ^aa , –CO ₂ R ^aa , –C(=O)N (R ^bb ) ₂ , –C(=NR ^bb )R ^aa , –C(=NR ^bb )OR ^aa , –C(=NR ^bb )N(R ^bb ) ₂ , –S(=O)R ^aa , – SO ₂ R ^aa , –Si(R ^aa ) ₃ , –P(R ^cc ) ₂ , –P(=O)(R ^aa ) ₂ and –P(=O)(OR ^cc ) ₂ , where R ^aa , R ^bb and R ^cc are as defined herein. Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in OrganiCSynthesis, TW Greene and PGM Wuts, 3rd Ed., John Wiley & Sons, 1999, incorporated herein by reference.

Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methyl Oxymethyl (SMOM), Benzyloxymethyl (BOM), p-Methoxybenzyloxymethyl (PMBM), (4-Methoxyphenoxy)methyl (p-AOM), Guaiac Glycanolmethyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2, 2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP ), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl Pyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidine-4 -yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuryl, tetrahydrothiofuryl, 2,3,3a,4,5,6,7,7a-octahydro-7,8 ,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1 -Methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2- Trimethylsilylethyl, 2-(phenylselenyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, Benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halogenated benzyl, 2,6-dichlorobenzyl , p-cyanobenzyl, p-phenylbenzyl, 2-pyridylmethyl, 4-pyridylmethyl, 3-methyl-2-pyridylmethyl N-oxide, diphenylmethyl, p,p' -Dinitrobenzhydryl, 5-dibenzocycloheptyl, triphenylmethyl, α-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, bis(p-methyl oxyphenyl)benzyl, tris(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4 ″-tris(4,5-dichlorophthalimidephenyl)methyl, 4,4′,4″tris(levulinylphenyl)methyl, 4,4′,4″-tri (Benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxy Phenyl)-1'-pyrenylmethyl, 9-anthracenyl, 9-(9-phenyl)anthracenyl, 9-(9-phenyl-10-oxo)anthracenyl, 1,3-benzo Dithiolan-2-yl, benzisothiazolyl S, S-dioxy, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl ( TIPS), Dimethylisopropylsilyl (IPDMS), Diethylisopropylsilyl (DEIPS), dimethyl-tert-hexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-di Tolylsilyl, triphenylsilyl, triphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, ethyl ester, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)valerate (levulinyl Dithioacetal), pivalate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-tris Methyl benzoate (mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(tri Methylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonyl)ethyl carbonate (Peoc), isobutyl carbonate ethylene carbonate, allyl carbonate, tert-butyl carbonate (BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl Carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2- Iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2 -(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro- 4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1, 1-Dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, O-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphoric diamide, alkyl N-phenylaminomethyl Ester, borate, dimethylthiophosphino, alkyl 2,4-dinitrophenyl sulfinate, sulfate, methanesulfonate (mesylate), benzylsulfonate and Tosylate (Ts).

In certain embodiments, the substituents present on the sulfur atom are sulfur protecting groups (also known as "mercapto protecting groups"). Sulfur protecting groups include but are not limited to -R ^aa , -N(R ^bb ) ₂ , -C(=O)SR ^aa , -C(=O)R ^aa , -CO ₂ R ^aa , -C(=O)N (R ^bb ) ₂ , –C(=NR ^bb )R ^aa , –C(=NR ^bb )OR ^aa , –C(=NR ^bb )N(R ^bb ) ₂ , –S(=O)R ^aa , – SO ₂ R ^aa , –Si(R ^aa ) ₃ , –P(R ^cc ) ₂ , –P(=O)(R ^aa ) ₂ and –P(=O)(OR ^cc ) ₂ , where R ^aa , R ^bb and R ^cc are as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in OrganiCSynthesis, TW Greene and PGM Wuts, 3rd Ed., John Wiley & Sons, 1999, incorporated herein by reference.

These and other exemplary substituents are described in more detail in the detailed description, embodiments and claims. The above exemplary list of substituents is not intended to limit the invention in any way.

C. Exemplary Synthesis of Cortistatin Analogs

Synthesis is first considered using compounds of formula (I) as starting materials. Oxidation (eg DDQ, _MnO2 ) of estrone (where ^R3 is _-CH3 ) or norestrone (where ^R3 is H) (I) provides compounds of formula (III). See, eg, Stephan et al., Steroid, 1995, 60, 809-811. Compounds of formula (III) are protected as acetals or ketals (e.g., by reaction with HX ^A R ^A or HX ^A R ^A - ^RA X ^A H, wherein two R ^A groups are linked, where R ^B1 and R ^B2 Each is independently -X ^A R ^A ), resulting in a mixture of (IV)-A and (IV)-B (eg 1:1 mixture). Exemplary conditions contemplated for protection include PTSA and ethylene glycol, PTSA and CH(OMe) ₃ , PTSA and CH(OEt) ₃ , and PTSA and 2,2-dimethyl-1,3-propanediol. The protected compound is then alkylated (eg methylated _{) using an alkylating agent (eg Me2SO4 and K2CO3, EtN(i-Pr)2 and TMS - diazomethane} ) to give (V )-A and (V)-B, wherein E is optionally substituted alkyl. See option 5.

Option 5

Scheme 6 provides other exemplary routes to provide compounds of formula (IV-B), for example, wherein ^R3 is _-CH3 . For example, as described in Scheme 6(A), compounds of formula (V)-B are obtained as racemic mixtures from 6-methoxy-1-tetralone in four steps. For stagger reactions see eg Saraber et al., Tetrahedron, 2006, 62, 1726-1742. For hydrogenation see eg Sugahara et al., Tetrahedron Lett, 1996, 37, 7403-7406. Scheme 6(B) shows a method for obtaining enantiomerically pure Torgov intermediates by chiral resolution. See eg Bucourt et al., J. Bull. Soc. Chim. Fr. (1967) 561-563. Scheme 6(C) provides an alternative method for the preparation of enantiomerically pure Torgov intermediates assisted by enzymatic reduction. See, eg, Gibian et al., Tetrahedron Lett. (1966) 7:2321-2330.

Option 6

Compounds of formulas (IV-A) and (IV-B) were in hand and subjected to epoxidation/epoxide ring opening/epoxidation reactions (e.g. MMPP, mCPBA) in a one-pot process to provide formula (IX-A ) and (IX-B), which in equilibrium, (IX-A) is the main compound. See Schemes 7A and 7B.

Option 7

Exposure of compounds of formula (IX-A) and (IX-B) to Birch reducing conditions (e.g. Li/NH and _t -BuOH, Na/NH and _t -BuOH) yields dearomatized compound (X) . C3 of the A ring is then protected as an acetal or ketal (e.g., by reaction with HX ^A R ^A or HX ^A R ^A - ^RA X ^A H, where two ^RA groups are linked, and where R ^B1 and R Each of ^B2 is independently -X ^A R ^A ) to obtain compound (XI). Exemplary protection conditions include PTSA and ethylene glycol, PTSA and CH(OMe) ₃ , PTSA and CH(OEt) ₃ , and PTSA and 2,2-dimethyl-1,3-propanediol. See Scheme 8.

Option 8

Compound (XI) is converted to compound of formula (XIII) by etherification (eg NBS, NIS, eg where X is Br or I). This compound is then oxidized (eg _SO3.Py/DMSO and triethylamine, IBX, (COCl) ₂ /DMSO and triethylamine) to give compounds of formula (XIV). This compound is then treated with a base (eg DBU, triethylamine) to provide compounds of formula (XV). This compound is then reduced (eg, NaBH ₄ and CeCl ₃ , L-selectride) to provide compounds of formula (XVI). See Scheme 9.

Option 9

Compounds of formula (XVI) are then treated with a cyclopropanation reagent (eg, ZnEt ₂ and ClCH ₂ I, ZnEt ₂ and CH ₂ I ₂ , Zn—Cu and CH ₂ I ₂ ) to provide compounds of formula (XVII). The alcohol of the cyclopropanation product is activated where LG ¹ is a sulfonyl group (for example, treatment of the alcohol with _Tf2O , MsCl to provide the activated alcohol where LG ¹ is Tf or Ms) and treated with a base (for example 2,6- Di-tert-butyl-4-methylpyridine, 2,6-lutidine, triethylamine) reaction to obtain the compound of formula (XX). See eg Magnus et al., Org. Lett. 2009, 11, 3938-3941. See Scheme 10.

Scheme 10

The protecting group on the D ring of compounds of formula (XX) is then deprotected under acidic conditions (eg PTSA and acetone/water, TFA/water) to provide ketone intermediates of formula (XXI). This product is treated with a compound of formula R _B1 -M (eg R _B1 -CeCl ₂ , R ^B1 -Mg) prepared from R ^B1 -X (eg R ^B1 -Br, R ^B1 -I) to give a compound of formula (XXII) Compounds wherein R ^B1 is a non-hydrogen group as defined herein. Compounds of formula (XXII) are activated (eg TFAA and pyridine, PhNCS and KH) to give compounds of formula (XXIII). Reduction of compounds of formula (XXIII) such as AIBN and _Bu3SnH provides compounds of formula (XXIV). For steps S14, S15 and S16, see eg Flyer et al., Nature. Chem. 2010, 2, 886-892., and Yamashita et al., J. Org. Chem. 2011, 76, 2408-2425. See Scheme 11A.

Compound (XXIV) can also be prepared from (XX) by conversion to an activated alcohol, wherein LG ² is sulfonyl (eg, treatment of the alcohol with Tf ₂ O, MsCl to provide the activated alcohol, wherein LG ² is Tf or Ms; by three fluoromethanesulfonation, such as KHMDS and PhNTf ₂ , LiHMDS and PhNTf ₂ , Tf ₂ O and 2,6-di-tert-butyl-4-methylpyridine), followed by palladium-catalyzed cross-coupling with _RB1 -M wherein M is a substituted boron (e.g. -B(R') ₂ , wherein each R' is -OR" or an alkyl, wherein the alkyl and R" are alkyl or may be joined to form a ring), to provide the formula (XXVI) compound. Exemplary palladium-catalyzed cross-coupling conditions include, but are not limited to, ^RB1 -B(pin), ^RB1- (9-BBN-H), ^RB1 -OBBD, or ^RB1 -B(cat), and Pd( PPh ₃ ) ₄ and Na ₂ CO ₃ or Pd(dppf)Cl ₂ and K ₃ PO ₄ ) (pin = pinacol; cat = catechol; OBBD = 9-oxa-10-bromobicyclo[3.3 .2] decane; 9-BBN-H=9-bromoheterobicyclo[3.3.1]nonane). See eg Nicolaou et al., J. Am. Chem. Soc. 2009, 131, 10587-10597. Hydrogenation of the C16-C17 double bond (eg Pd/C and _H2 , Raney nickel and _H2 ) yields compounds of formula (XXIV). See Scheme 11B.

Scenario 11.

Either compound of formula (XXVI) or (XXIV) can then be deprotected (e.g., PTSA and acetone/water, TFA/water, HCl) and the resulting ketone can be trapped as an enolate followed by oxidation of the double bond or Amination, or reaction of the double bond with the electrophilic carbon C( ^RA ) ₃ -LG (where LG is the leaving group) to provide substituted ketone products where R ⁵ is -OR ^A , -OC(=O)R ^A , -OC(=O)OR ^A , -OC(=O)N(R ^A ) ₂ , -OS(=O) ₂ R ^A , -N ₃ , -N(R ^A ) ₂ , -NR ^A C (=O)R ^A , -NR ^A C(=O)OR ^A , -NR ^A C(=O)N(R ^A ) ₂ , -NR ^A S(=O) ₂ R ^A or –C(R ^A ) ₃ . See Schemes 12A and 12B. Exemplary conditions considered for enolate trapping include bases (e.g., lithium diisopropylamide (LDA)) and bases in which P is _a silyl group and LG is a leaving group (e.g., trimethylsilyl Chloride) combination of capture reagents P ₁ -LG.

Exemplary oxidation conditions such as -OR ^A , -OC(=O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ or -OS(=O) ₂ Installation of the ^RA group at the R position involves treatment of the trapped enolate with an oxidizing agent such as m ^- chloroperoxybenzoic acid (MCPBA), ^MoOOPh , or DMSO to provide a substituted ketone wherein R is -OH, followed by optional protection, e.g. By using the formula R ^A -LG, LG-C(=O) ^RA , LG-C(=O)OR ^A , LG-C(=O)N( ^RA ) ₂ or Compounds of LG-S(=O) _2RA Treatment of compounds wherein R5 is -OH affords compounds wherein R5 is ^-ORA (where ^RA is ^a non ^- hydrogen group), -OC( ⁼ O) ^RA , -OC(=O)OR ^A , -OC(=O)N( ^RA ) ₂ or -OS(=O) ₂ R ^A .

Exemplary amination conditions, such as _-N3 , -N( ^RA ) ₂ , ^-NRAC (=O) ^RA , ^-NRAC (=O) ^ORA , ^-NRAC (=O) N( ^RA ) ₂ or -NR ^A S(=O) ₂ ^RA group installed at the R ⁵ position, including treatment with a compound N ₃ -LG in which LG is the leaving group (e.g. triazide) is Trapped _enolates to afford substituted ketones where R5 is ^-N3 . Substituted ketones wherein R5 is _-N3 can be treated with a reducing agent such as ^PPH3 to provide compounds wherein R5 is _-NH2 , followed by optional protection, for example by using a ^formula _R wherein LG is a leaving group Compounds of ^A -LG, LG-C(=O) ^RA , LG-C(=O)OR ^A , LG-C(=O)N( ^RA ) ₂ or LG- ^S (=O) _2RA Compounds wherein R is -NH are treated to provide wherein R is -N( ^RA _{) 2} ⁽ wherein at least one RA is ^a non _- hydrogen group), ^-NRAC ( ⁼ O) ^RA , -NR ^A compound of A C(=O)OR ^A , -NR ^A C(=O)N( ^RA ) ₂ or -NR ^A S(=O) ₂ R ^A .

Scheme 12

The ketone compound as provided in Schemes 12(A) and 12(B) can then be treated with ^an amine of _formula H2NR1 to form the condensation product imine as described in step S22. The ketone compound can also be treated with an amine of formula HNR ¹ R ² or a salt thereof under reductive amination conditions to provide the aminated product, as described in step S23. Exemplary reductive amination conditions include, but are not limited to, _NaCNBH3 , NaCN(9BBN)H, or NaBH(OAc) ₃ at acidic pH (eg, pH 3). As shown in step S25, the aminated product can be further oxidized to the corresponding N-oxide. Exemplary oxidizing conditions include, but are not limited to, H2O2, _mCPBA , or _DMDO . See Schemes 13A to 13D.

Scheme 13

^Ketones _{can also} be ^converted to _formula Compounds of (XXV-i). The conditions of the following steps to obtain the compounds of formulas (XXV-i), (XXV-iv) and (XXV-v) are the same as above. See Scheme 14.

Scheme 14

The ketone compounds provided in Scheme 14 can then be converted to the corresponding imines, amines and N-oxides as previously described. See Schemes 15A and 15B.

Scheme 15

Monoketones (XXI) can be reductively aminated with HNR ^B4 R ^B5 (e.g. 1,2,3,4-tetrahydro-[2,7]naphthyridine) under previously described conditions to afford formula (XXVII) compound. Compound (XXVII) can be converted to the corresponding imine, amine and N-oxide as previously described. See Schemes 16(A) and 16(B).

Scheme 16

Ketones can be further manipulated synthetically to provide other compounds of interest. For example, a ketone can be reduced in the presence of a reducing agent (as shown in step S26) to provide a C-3 hydroxylated compound. See Scheme 17(A) and (B). Exemplary reducing agents include L-selectride, K-selectride, diisobutylaluminum hydride (DIBALH), and lithium aluminum hydride (LAH). In addition, various reducing agents will preferentially produce one C-3 hydroxylated compound as the major isomer relative to another C-3 hydroxylated compound, for example, using the L-selectride beta isomer preferentially produced as the major isomer , while the use of lithium aluminum hydride (LAH) alpha isomer is preferably produced as the major isomer.

Program 17

The C-3 hydroxylated compound can then be activated (e.g., by reacting under Mitsunobu reaction conditions (e.g., with HOC(=O) ^RA , diethyl azodicarboxylate, Ester (DEAD) or diisopropyl azodicarboxylate (DIAD) and PPH ₃ ) are substituted with a group of formula -C(=O) ^RA with the group LG-C(=O) ^RA (wherein LG is Leaving group) reaction), followed by treatment with an amine of formula NHR ¹ R ² to provide the compound of formula (XXIV) with inverted C3 stereochemistry as the major isomer (as described in step S28). See Scheme 17(A) and (B). Alternatively, a C-3 hydroxylated compound of formula (XXX1) can be treated with a base and a compound of ^formula RO-LG wherein LG is the leaving group to provide the protected C3-hydroxyl compound, the major isomer of C3 Stereochemistry is preserved (as described in step S27).

Ketones of Ring A can be further manipulated synthetically to provide compounds as described herein. Taking the ketone of formula (XXIV-i) as an example, the ketone can be converted to a free oxime (see e.g. Scheme 18) or a substituted oxime, where R ^O is a non-hydrogen group (see e.g. Scheme 19), followed by a Beckmann rearrangement Conversion to provide the desired lactam product. For example, free oximes can be generated from ketones upon treatment with hydroxylamine _NH2OH and can directly provide lactam products under suitable rearrangement conditions (eg acidic conditions eg _H2SO4 , HCl, AcOH ₎ . See, eg, Scheme 18.

Program 18.

Alternatively, substituted oximes where R ^O is a non-hydrogen group can be generated from ketones in one step (S26), for example by treatment with substituted hydroxylamines NH ₂ OR ^O , where R ^O is a non-hydrogen group, or can be generated via a two-step method (S23 ) and (S27) are generated, for example, by treatment first with hydroxylamine NH2OH and then with a compound of _formula RO-LG, where ^RO is a non- ^hydrogen group and LG is a leaving group. See, eg, Scheme 18. Exemplary leaving groups (LG) include halogen (eg, chlorine, bromine, iodine) and _-OSO2R ^aa , wherein R ^aa is as defined herein. The group -OSO ₂ R ^aa includes leaving groups such as tosyl, mesyl and benzenesulfonyl, where R ^aa is optionally substituted alkyl (eg, -CH ₃ ) or optionally substituted aryl ( such as phenyl, tolyl). Exemplary compounds of formula R ^O -LG include LG-C(=O) ^RA , LG-C(=O)OR ^A , LG-C(=O)N( ^RA ) ₂ , LG-S(=O ) ₂ R ^A , LG–Si(R ^A ) ₃ , LG–P(=O)(R ^A ) ₂ , LG–P(=O)(OR ^A ) ₂ , LG–P(=O)(NR ^A ) ₂ , LG–P(=O) ₂ R ^A , LG–P(=O) ₂ (OR ^A or LG–P(=O) ₂ N( ^RA ) ₂ , where LG is as defined herein. Specifically consider Compounds of the formula LG-S(=O) ₂ R ^A include Cl-S(=O) ₂ CH ₃ (MsCl), Cl-S(=O) ₂ C ₆ H ₄ -( _p CH ₃ )(TsCl) and Cl—S(=O) ₂ C ₆ H ₅ (BsCl). Substituted oximes can directly afford lactam products under suitable rearrangement conditions (eg, acidic conditions such as H ₂ SO ₄ , HCl, AcOH).

Program 19

Alternatively, ketones can be reduced under Wolff-Kishner reducing conditions (as described in step S30) to provide compounds of formula (G1') and (G1"). See Scheme 20. Exemplary Wolff-Kishner conditions are described in Furrow, M.E.; Myers, A.G. (2004). "Practical Procedures for the Preparation of N-tert-Butyldimethylsilylhydrazones and Their Use in Modified Wolff-Kishner Reductions and in the Synthesis of Vinyl Halides and gem-Dihalides". Journal of the American Chemical Society 126(17) : 5436–5445, incorporated herein by reference.

Program 20

As understood herein, the oxime produced by the above reaction may comprise a single oxime C3 isomer product or a mixture of two oxime C3 isomer products. It is also generally understood that Beckmann rearrangements proceed via trans [1,2]-shift. Thus, in any given reaction, a mixture of lactam products is expected to be produced, with one lactam being the predominant product.

The lactam product can then be reduced to the azepine product using various conditions, for example using hydrides (e.g. lithium aluminum hydride), Clemmenson reduction (e.g. Zn(Hg)/HCl) and Wolff Kishner reduction ( Examples include hydrazine and bases such as KOH). See, eg, Scheme 21.

Scheme 21

Compounds of formula (E1') or (E1") where X is chlorine, bromine or iodine can be synthesized by hydrolysis of the lactam to the carboxylic acid followed by decarboxylation and halogenation followed by cyclization. See eg Schemes 22A and 22B.

Plan 22A

Plan 22B

Compounds of formula (E2') or (E2") can entrap ketones of formula (B*') or (B*") (where R is a non- ^hydrogen group as defined herein), alkenyl moieties via enols Synthesized by oxidative cleavage of , formation of an acyl azide followed by a Curtius rearrangement to provide an amino moiety, which is subsequently cyclized to provide a lactam, reduced to a piperazinyl product wherein R ^N is hydrogen, which can optionally be synthesized by Non-hydrogen group R ^N protection. See, eg, Scheme 23A or 23B.

Plan 23A

Plan 23B

As used herein, "major isomer" refers to an isomer produced in excess of another isomer, i.e. greater than 50% of the sum of the two isomers produced by the reaction, for example greater than the 60%, 70%, 80%, 90% or 95% of the sum of the two isomers.

D. Representative Synthesis of Cortistatin Analogs

Materials and Instruments:

All reactions were performed in flame-dried glassware under a positive pressure of argon unless otherwise stated. Flash column chromatography employs silica gel 60 (40-63 μm, Whatman) as described by Still et al., J. Org. Chem. 1978, 43, 2923-2925.

Commercially available reagents and solvents were used as received with the following exceptions: Tetrahydrofuran (THF), dichloromethane ( _{CH2Cl2 )} were degassed with argon, and allowed to pass through as described in Pangborn et al., Organometallics 1996, 15 , 1518–1520 described a solvent purification system on an alumina column (designed by JCMeyer of Glass Contour). Pyridine and triethylamine were distilled from calcium hydride before use. The diatomaceous earth used was purchased from JT Baker 545. The molarity of the n-butyllithium solution was determined by titration (average of three determinations) using 1,10-phenanthroline as an indicator.

¹ H NMR spectra were recorded on a Varian INOVA-600 or Varian INOVA-500 spectrometer. Proton chemical shifts are reported in parts per million (δ scale) and use residual undeuterated solvent as an internal reference (CDCl ₃ : δ 7.26 (CHCl ₃ ), C ₆ D ₆ : δ 7.15 (C ₆ D ₅ H)) for calibration. Data for ¹ H NMR spectroscopy are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, app = pronounced, or combinations thereof. ¹³ C NMR spectra were recorded on a Varian INOVA-500 spectrometer. High-resolution mass spectra (HRMS) were obtained from Harvard University Mass Spectrometry Laboratory, where electrospray ionization (ESI) mass spectrometry (MS) experiments were performed on an Agilent 6210TOF LC/MS instrument.

Example S1. Synthesis of Ketone Starting Materials

Scheme 1-1

Scheme 1-2.

Route 1: Synthesis of 8,9-unsaturated methoxyketene (compound 1) from 6-methoxy-1-tetralone

The Grignard reaction was carried out with 20.0 g (113 mmol, 1.00 equiv) of 6-methoxy-1-tetralone and the product was used without flash chromatography. See eg Saraber et al., Tetrahedron 2006, 62, 1726-1742. To a solution of the Grignard reaction product and 2-methyl-1,3-pentadienone (12.8 g, 114 mmol, 1.01 eq) in xylene (140 mL) was added AcOH (64.6 mL, 1.13 mol, 10.0 eq) and The reaction mixture was heated to reflux. After 2 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. A 1:1 mixture of toluene and diethyl ether was added to dissolve the solid residue and the mixture was filtered. The filtrate was washed sequentially with saturated NaHCO ₃ solution (200 mL) and brine, dried over MgSO ₄ , and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent: 20:1:1 hexane:EtOAc:DCM) to give the Torgov diene. Spectral data were consistent with previously reported. See, eg Soorukram, D.; Knochel, P. Org. Lett. 2007, 9, 1021-1023. Based on methods known from the literature, the Torgov diene was converted to the 8,9-unsaturated methoxyketene compound 1 (15.0 g, 47% over 3 steps). See eg Sugahara et al., Tetrahedron Lett. 1996, 37, 7403-7406.

Route 1: Synthesis of 8,9-unsaturated methoxyethylene ketal (compound 2)

To a solution of compound 1 (15.0 g, 53.1 mmol, 1.0 equiv) in benzene (215 mL) and ethylene glycol (72 mL) was added oxalic acid (2.30 g, 12.1 mmol, 0.22 equiv). The reaction mixture was warmed to reflux and water was captured by a Dean-Stark apparatus. After 16 hours, the reaction was cooled to room temperature and saturated NaHCO ₃ solution (150 mL) was added. The organic and aqueous layers were separated, and the aqueous phase was extracted with ethyl acetate (2 x 200 mL). The combined organic phases were washed with brine (150 mL) and dried over Na ₂ SO ₄ . The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (silica gel, eluent: 15:1 hexane:EtOAc) to give 8,9-unsaturated methoxyethylene ketal compound 2 (15.5 g, 89% ). ¹ H NMR (500MHz, CDCl ₃ ) shift = 7.13 (d, J = 8.3Hz, 1H), 6.73-6.67 (m, 2H), 4.05-3.85 (m, 4H), 3.79 (s, 3H), 2.82- 2.65(m,2H),2.52-2.45(m,2H),2.23-2.17(m,2H),2.14(ddd,J=2.2,11.6,14.0Hz,1H),1.99-1.82(m,4H), 1.64(td,J=4.2,12.2Hz,1H), 1.49(dq,J=6.8,11.6Hz,1H),0.86(s,3H).HRMS(ESI)(m/z)C ₂₁ H ₂₇ O ₃ [M+H] ⁺ : calculated value 327.1955, measured value 327.1947.

Route 1: Synthesis of epoxy alcohols 3 and 3a

A solution of 8,9-unsaturated ethylene ketal 2 (1.63 g, 5.00 mmol, 1.0 equiv) in CHCl ₃ (50 mL) was cooled to 0° C., mCPBA (77% max, 2.46 g, 11.0 mmol, 2.2 equiv. ). The reaction mixture was stirred at 0 °C for 10 minutes and allowed to warm to room temperature. After another 50 min, ₁₀ % Na2S2O3 solution (40 mL ₎ followed by saturated _NaHCO3 solution (40 mL ₎ was added sequentially. The organic and aqueous layers were separated, and the aqueous phase was extracted with dichloromethane (3 x 50 mL). The combined organic phases were washed with brine (50 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent: 3:1 → 1:1 hexane:EtOAc) to afford epoxy alcohols 3 and 3a (1.40 g, 75%). 3 and 3a are in equilibrium in any solvent, mostly 3. Analysis of H NMR of epoxy alcohol 3. In continuation, cortistatin analogs (12, 13, 14A, 14B, 15B, 16B and 17B) were applied to biological experiments as racemic mixtures constructed from 6-methoxy-1-tetralone .

¹ H NMR (500MHz, CDCl ₃ ) Shift = 7.77 (d, J = 8.3Hz, 1H), 6.76 (dd, J = 2.0, 8.3Hz, 1H), 6.63 (d, J = 2.0Hz, 1H), 4.78 (dd, J=7.8,9.8Hz,1H),3.95-3.87(m,4H),3.78(s,3H),2.84(dt,J=5.9,14.4Hz,1H),2.49(dd,J=4.4 ,15.1Hz,1H),2.36-2.29(m,1H),2.26(dd,J=5.9,14.2Hz,2H),2.06(t,J=11.7Hz,1H),1.97(dd,J=7.3, 12.2Hz, 1H), 1.94-1.88(m, 2H), 1.75(dt, J=5.4, 14.2Hz, 1H), 1.63-1.53(m, 1H), 1.46(t, J=11.0Hz, 1H), 0.75(s,3H).HRMS(ESI)(m/z)C ₂₁ H ₂₇ O ₅ [M+H] ⁺ : Calculated 359.1853, Found 359.1852.

Route 2: Synthesis of 8,9 and 9,11-unsaturated methoxyethylene ketal compounds 2 and 4

DDQ oxidation was accomplished with 22.0 g (81.4 mmol, 1.0 equiv) of estrone and the product was used without flash chromatography. See eg Stephan et al., Steroid. 1995, 60, 809-811. To a solution of 9,11-unsaturated estrone in benzene (375 mL) was added ethylene glycol (110 mL, 1.99 mol, 24.4 equiv) and PTSA (3.00 g, 16.3 mmol, 0.20 equiv). The reaction mixture was warmed to reflux and water was captured with a Dean-Stark apparatus. After 18 hours, the reaction was cooled to room temperature, and saturated NaHCO ₃ solution (300 mL) was added. The aqueous phase was extracted with ethyl acetate (2 x 300 mL), and the combined organic phases were washed with brine (200 mL). The organic phase was dried ( _{Na2SO4 )} and the solvent was evaporated under reduced pressure. The product was used in the next step without further purification.

Vinyl ketal (a mixture of 8,9 and 9,11-unsaturated regioisomers) was dissolved in acetone (420 mL), and K ₂ CO ₃ (22.5 g, 163 mmol, 2.00 eq) was added. _{Me2SO4 (} 9.30 mL, 97.6 mmol, 1.20 equiv) was then added and the reaction mixture was warmed to reflux. After 18 hours, the reaction was allowed to cool to room temperature and the acetone was evaporated. 2M NaOH solution (300 mL) was added and the aqueous phase was extracted with ethyl acetate (2 x 300 mL). The combined organic phases were dried ( _{Na2SO4 )} and the solvent was evaporated under reduced pressure. Purification of the residue by flash chromatography (silica gel, eluent: 15:1 hexane:EtOAc) afforded 8,9 and 9,11-unsaturated methoxyethylene ketal compounds 2 and 4 (16.3 g in three steps 61%, ~4:5 mixture of 8,9-unsaturated regioisomer:9,11-unsaturated regioisomer).

For the 9,11-unsaturated isomer, only distinguishable peaks can be assigned: ¹ H NMR (500 MHz, CDCl ₃ ) shift = 7.53 (d, J = 8.8 Hz, 1H), 6.60 (d, J = 2.0 Hz ,1H),6.13(td,J=2.6,5.0Hz,1H),3.79(s,3H),2.59(td,J=3.2,17.6Hz,1H),2.09-2.00(m,3H),1.45- 1.33 (m, 2H), 0.90 (s, 3H). HRMS (ESI) (m/z) C ₂₁ H ₂₇ O ₃ [M+H] ⁺ : calculated 327.1955, found 327.1951.

Route 2: Epoxy Alcohol Compounds 3 and 3a

To a solution of a mixture of 8,9 and 9,11-unsaturated ethylene ketal compounds 2 and 4 (15.7 g, 48.1 mmol, 1.00 equiv) in dichloromethane (700 mL) was added monoperoxyphthalic acid hexahydrate Magnesium (68.4 g, 111 mmol, 2.30 equiv) and water (4.8 mL). The reaction mixture was stirred at room temperature for ₂₀ h, then quenched with a mixture of ₁₀ % aqueous Na2S2O3 ( ₃₀₀ mL) and saturated _NaHCO3 solution (300 mL). The organic and aqueous layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 500 mL). The combined organic phases were washed with brine (300 mL) and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (silica gel, eluent: 3:1→2:1 hexane:EtOAc) to afford epoxy alcohols 3 and 3a (8.60 g, 50%). Spectral data are consistent with epoxy alcohols 3 and 3a constructed from 8,9-unsaturated methoxyethylene ketal 2.

Synthesis of Diol Compound 5

Ammonia gas was condensed (240 mL), and Li (3.90 g, 565 mmol, 25.0 equiv) was added to liquid ammonia at -78 °C. After stirring for 30 minutes, epoxy alcohols 3 and 3a (8.10 g, 22.6 mmol, 1.0 equiv) in THF (110 mL) were cannulated at this temperature and stirred for an additional 1.5 hours. A mixture of t-BuOH (32 mL) and THF (16 mL) was added to the reaction mixture at -78°C and stirred at this temperature for another 20 minutes. A mixture of t-BuOH (92 mL) and THF (38 mL) was added at -78°C, followed by benzene (50 mL) and water (50 mL), the flask was opened to gently evaporate the liquid ammonia by removing the cooling bath. Water (200 mL) was added and the aqueous phase was extracted with ethyl acetate (2 x 250 mL). The combined organic phases were washed with brine (150 mL), dried (Na ₂ SO ₄ ), and concentrated under reduced pressure. The product was used in the next step without further purification.

To a solution of the Birch reduction product in THF (300 mL) and ethylene glycol (75 mL) was added PTSA (430 mg, 2.26 mmol, 0.10 equiv). The reaction mixture was stirred at room temperature for 30 minutes, and saturated NaHCO ₃ solution (200 mL) was added. The organic and aqueous layers were separated, and the aqueous phase was extracted with ethyl acetate (4 x 250 mL). The combined organic phases were washed with brine (200 mL) and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure and the residue was purified by flash chromatography (silica gel, eluent: 4:1 hexane:EtOAc→100% EtOAc→10:1 EtOAc:MeOH) to afford diol 5 (4.60 g, 52 %).

¹ H NMR (500MHz, C ₆ D ₆ ) Shift = 3.67-3.42(m, 9H), 3.25-3.14(m, 1H), 2.40(dd, J=5.9, 13.2Hz, 1H), 2.31(br.s ,2H),2.23-2.09(m,2H),2.03(t,J=10.7Hz,1H),1.97-1.90(m,2H),1.89(dd,J=8.3,14.2Hz,1H),1.85- 1.75(m,4H),1.66-1.50(m,4H),1.00(s,3H).HRMS(ESI)(m/z)C ₂₂ H ₃₂ NaO ₆ [M+Na] ⁺ : calculated value 415.2091, measured Value 415.2076.

Scenarios 1-3. Optimized route 2

Keto compound 3b

To a solution of estrone (195 g, 721 mmol, 1.00 equiv) in DMSO (2.8 L) was added KOH pellets (85% technical grade, 162 g, 2.45 mol, 3.40 equiv) and _CH3I (89.8 mL, 1.44 mol, 2.00 equiv). The reaction mixture was stirred at room temperature for 3.5 hours, and distilled water (2 L) was slowly added at 0°C. The aqueous layer was extracted with dichloromethane (3 x 1.5 L) and the combined organic layers were washed with brine (1.5 L). The organic layer was concentrated under nitrogen flow to give white crystals, which were washed with cold methanol. 180 g of the crude mixture was used in the next step without further purification.

To a solution of the crude mixture (100 g, 352 mmol, 1.00 equiv) in methanol (750 mL) and dichloromethane (750 mL) was added NaHCO ₃ (93.8 g, 1.05 mmol, 3.00 equiv). DDQ (120 g, 527 mmol, 1.50 equiv) was added in four portions at ₅ min intervals and the reaction mixture was stirred for ₂ h, then quenched with ₁₀ % aqueous Na2S2O3 (500 mL). The reaction flask was stirred for an additional 30 minutes, filtered through celite and washed with chloroform. 2M NaOH solution (500 mL) was added, the organic and aqueous layers were separated, and the aqueous phase was extracted with chloroform (3 x 700 mL). The combined organic phases were washed with brine (700 mL) and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure and 89 g of the crude mixture was used in the next step without further purification. The DDQ oxidation step was performed in two batches.

To a solution of the crude mixture (151 g, 480 mmol, 1.00 equiv) in benzene (2 L) was added ethylene glycol (268 mL, 4.80 mol, 10 equiv) and PTSA (27.4 g, 144 mmol, 0.30 equiv). The reaction mixture was warmed to reflux and water was captured with a Dean-Stark apparatus. After 36 hours, the reaction was cooled to room temperature, and saturated NaHCO ₃ solution (1 L) was added. The aqueous phase was extracted with ethyl acetate (3 x 500 mL), and the combined organic phases were washed with brine (1 L). The organic phase was dried ( _{Na2SO4 )} and the solvent was evaporated under reduced pressure. 170 g of crude product was used in the next step without further purification.

To a solution of the crude mixture (480 mmol, 1.00 eq) in dichloromethane (2.5 L) was added magnesium monoperoxyphthalate hexahydrate (~80% technical grade, 683 g, 1.10 mol, 2.30 eq) and water (50 mL ). The reaction mixture was stirred at room temperature for 16 hours, then filtered through a pad of celite. Saturated NaHCO ₃ solution (1.5 L) was added to the filtrate, the organic and aqueous layers were separated, and the aqueous phase was extracted with dichloromethane (3×1.4 L). The combined organic phases were washed with brine (1.4 L) and dried (Na ₂ SO ₄ ). The solvent was evaporated under reduced pressure and the crude mixture was used in the next step without further purification.

To a solution of the crude mixture (480 mmol, 1.00 equiv) in 1,2-dichloroethane (2 L) was added NaBH ₃ CN (60.3 g, 960 mmol, 2.00 equiv) and AcOH (55 mL, 960 mmol, 2.00 equiv) at room temperature ). After 2.5 hours, saturated NaHCO ₃ solution (1.4 L) was added and the organic and aqueous layers were separated. The aqueous phase was extracted with dichloromethane (3 x 1.4 L). The combined organic phases were washed with brine (1.5 L), dried _{over Na2SO4} , and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent: 2:1 hexane:EtOAc→1:1→1:2→1:3→100% EtOAc) to provide compound 3b (75 g, 29% for 5 steps ).

¹ H NMR (500MHz, CDCl ₃ ) shift = 7.21 (d, J = 8.8Hz, 1H), 6.75 (dd, J = 2.4, 8.3Hz, 1H), 6.72 (d, J = 2.4Hz, 1H), 3.98 -3.82(m,4H),3.79(s,3H),3.80-3.76(m,1H),3.54(dt,J＝4.4,10.5Hz,1H),3.03-2.91(m,1H),2.81(td ,J=4.4,18.1Hz,1H),2.33(d,J=9.8Hz,1H),2.23(dd,J=6.8,13.2Hz,1H),2.09-1.98(m,1H),1.90(ddd, J=5.9,9.8,14.6Hz,1H),1.85(dd,J=4.6,9.5Hz,2H),1.82-1.77(m,1H),1.77-1.70(m,1H),1.65(dq,J= 6.3,12.7Hz,1H),1.02(s,3H).HRMS(ESI)(m/z)C ₂₁ H ₂₈ O ₅ [M+H] ⁺ : calculated value 361.2010, measured value 361.2022.

Diol compound 5

Compound 3b (40 _g , 111 mmol, 1.00 equivalent) in THF (500 mL) and allowed to warm to 0°C. The reaction was followed by MS. After stirring at 0 °C for 7 h, the reaction was quenched by the slow addition of MeOH (150 mL) and _H2O (250 mL) and allowed to warm to room temperature. After the solution was decanted to separate the silica gel, EtOAc (1 L) was added and the organic and aqueous layers were separated. The aqueous phase was extracted with EtOAc (3 x 500ml). The combined organic phases were washed with brine (2 x 1 L), dried _{over Na2SO4} , and concentrated under reduced pressure. The product was used without further purification. Ketalization conditions were the same as described for compound 3b to afford compound 5 (32 g, 74% for two steps).

Allyl alcohol 7

To a solution of diol 7 in dichloromethane (230 mL) (7.1 g, 18.1 mmol, 1.00 equiv) was added NBS (3.54 g, 19.9 mmol, 1.10 equiv) in one portion at 10 °C, and the reaction mixture was warmed to room temperature . The reaction was monitored by TLC (about 2 hours to completion). Once the reaction was complete, the reaction mixture was cooled to -40 °C and triethylamine (30.3 mL, 217 mmol, 12.0 equiv) was added. SO3 _- Py (28.8 g, 181 mmol, 10.0 equiv) pre-stirred in DMSO (200 mL) for 20 min at room temperature was added to the reaction mixture at -40 °C, which was then slowly warmed to room temperature. After 3 hours, saturated _NH4Cl solution (200 mL) was added and the reaction was allowed to warm to room temperature. The organic and aqueous layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 350 mL). The combined organic phases were washed with brine (350 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude mixture was used without further purification.

The crude mixture was dissolved in dichloromethane (600 mL), the reaction mixture was cooled to -40 °C, then DBU (6.76 mL, 45.3 mmol, 2.50 equiv) was added slowly. After 15 minutes, saturated _NH4Cl solution (200 mL) was added and the reaction was allowed to warm to room temperature. The organic and aqueous layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 200 mL). The combined organic phases were washed with brine (150 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure.

To a solution of the crude mixture (6.50 g, 16.7 mmol, 1.00 equiv) in MeOH (250 mL) and THF (30 mL) at room temperature was added CeCl ₃ ·7H ₂ O (18.7 g, 50.2 mmol, 3.00 equiv) ( 18.7 g, 50.2 mmol, 3.00 equiv). After stirring for 5 minutes, the reaction was cooled to -20 °C, and then NaBH4 ₍ 1.26 g, 33.4 mmol, 2.00 equiv) was added. After 30 min, saturated _NH4Cl solution (100 mL) and water (100 mL) were added and it was allowed to warm to room temperature. The aqueous phase was extracted with ethyl acetate (3 x 250 mL), the combined organic phases were washed with brine (200 mL), dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent: 20:1 DCM:MeOH) to afford allyl alcohol 7 (4.20 g, 60% for 3 steps).

¹ H NMR (500MHz, C ₆ D ₆ ) Shift=4.39-4.30(m,1H),3.58-3.36(m,8H),3.22(dd,J=3.7,16.4Hz,1H),2.94(dd,J =7.1,12.5Hz,1H),2.66(d,J=13.2Hz,1H),2.49-2.41(m,1H),2.39(dd,J=2.2,12.9Hz,1H),2.07-1.99(m, 1H), 1.96-1.79(m, 6H), 1.73(br.s, 3H), 1.66-1.57(m, 1H), 1.15-1.07(m, 1H), 0.86(s, 3H); ¹³ C NMR ( 500MHz, C ₆ D ₆ ) displacement = 140.6, 139.1, 118.7, 109.5, 88.3, 86.2, 67.1, 65.4, 64.6, 64.2, 47.9, 46.5, 41.3, 40.9, 34.7, 34.2, 33.9, 30.0, 20.4, 19.8, 15.6 ; HRMS (ESI) (m/z) C ₂₂ H ₃₀ NaO ₆ [M+Na] ⁺ : calculated 413.1935, found 413.1942.

Cyclopropane 8

To a solution of ClCH ₂ I (5.74 mL, 78.9 mmol, 4.00 equiv) in 1,2-dichloroethane (400 mL) was added Et ₂ Zn in diethyl ether (1M, 39.4 mL, 39.4 mmol, 2.00 equiv) at -10°C ) in the solution. After stirring for 5 minutes, allyl alcohol 7 (7.70 g, 19.7 mmol, 1.00 equiv) in 1,2-dichloroethane (200 mL) was added to the reaction flask at -10 °C. After 30 minutes, the reaction was quenched by saturated _NH4Cl solution (300 mL) and allowed to warm to room temperature. The organic and aqueous layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 350 mL). The combined organic phases were washed with brine (300 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent: 2:1 → 1:1 hexane:EtOAc) to afford cyclopropane 8 (6.93 g, 87%).

1H NMR (500MHz, C6D6) shift = 3.92 (dd, J = 3.7, 11.0Hz, 1H), 3.51-3.40 (m, 8H), 2.72 (dd, J = 7.1, 12.9Hz, 1H), 2.39 (dd, J＝5.4, 17.6Hz, 1H), 2.38(d, J＝12.2Hz, 1H), 2.15(d, J＝12.2Hz, 1H), 2.12(dt, J＝4.9, 12.2Hz, 1H), 2.02( ddd, J＝2.9, 11.2, 14.6Hz, 1H), 1.92-1.82(m, 3H), 1.82-1.73(m, 2H), 1.69-1.54(m, 5H), 1.52(dd, J＝6.1, 12.0 Hz,1H),1.49-1.44(m,1H),0.98(s,3H),0.86(d,J=2.4Hz,1H),0.15(d,J=2.9Hz,1H);13C NMR(500MHz, C6D6) Displacement = 118.5, 110.4, 85.4, 84.0, 65.3, 64.9, 64.7, 64.6, 64.1, 48.1, 45.4, 41.5, 40.0, 39.9, 35.4, 34.8, 33.7, 32.7, 29.1, 22.1, 19.3, 16.5, 4.0; HRMS(ESI)(m/z) C ₂₃ H ₃₂ NaO ₆ [M+Na] ⁺ : Calculated 427.2091, Found 427.2088.

Oxabicyclo[3.2.1]octene skeleton 9

Cyclopropane 8 (6.90 g, 17.1 mmol, 1.00 equiv) and 2,6-di-tert-butyl-4-picoline (12.3 g, 59.7 mmol, 3.50 equiv) were azeotropically dried with benzene and dissolved in dichloromethane (330mL). join in Molecular sieves (8.6 g), the reaction flask was cooled to 0°C. A solution of trifluoromethanesulfonic anhydride in dichloromethane (1M, 34.1 mL, 34.1 mmol, 2.00 equiv) was added dropwise, the ice bath was removed, and the reaction flask was allowed to warm to room temperature. After 2 hours, the reaction was quenched with triethylamine (55 mL) and filtered through a pad of celite. Sat. NaHCO ₃ solution (300 mL) was added and the aqueous phase was extracted with dichloromethane (2×350 mL). The combined organic phases were washed with brine (300 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, eluent: 9:1→4:1 benzene:ether) to afford the oxabicyclo[3.2.1]octene core skeleton 9 (3.76 g, 57%).

¹ H NMR (500MHz, CDCl ₃ ) Shifts = 5.73(s, 1H), 5.29-5.26(m, 1H), 4.04-3.76(m, 8H), 2.58-2.50(m, 1H), 2.46(t, J =15.1Hz, 1H), 2.31-2.24(m, 2H), 2.19(t, J=11.2Hz, 1H), 2.09(d, J=13.2Hz, 1H), 1.99(dt, J=4.4, 13.2Hz ,1H),1.94(dd,J=2.4,13.2Hz,1H),1.91-1.84(m,1H),1.83-1.71(m,3H),1.71-1.53(m,5H),0.88(s,3H ); ¹³ C NMR (500MHz, CDCl ₃ ) shift = 140.6, 139.9, 119.9, 119.8, 118.5, 108.9, 81.5, 80.0, 65.2, 64.6, 64.5, 64.2, 46.2, 45.9, 42.4, 39.8, 34.0, 33.2, 32.4 , 31.1, 28.0, 18.5, 17.0; HRMS (ESI) (m/z) C ₂₃ H ₃₁ O ₅ [M+H] ⁺ : calculated value 387.2166, found value 387.2180.

monoketone 10

To a solution of oxabicyclo[3.2.1]octene core skeleton 9 (3.24 g, 8.38 mmol, 1.00 equiv) in acetone (400 mL) and water (100 mL) was added PTSA (797 mg, 4.19 mmol, 0.50 equiv), and The reaction mixture was stirred for 3 days. Sat. NaHCO ₃ solution (210 mL) and ethyl acetate (300 mL) were sequentially added to the reaction. The layers were separated and the aqueous layer was extracted with ethyl acetate (2 x 200 mL). The organic layers were combined, washed with brine (150 mL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The resulting residue was then purified by flash chromatography (silica gel, eluent: 4:1 hexane:EtOAc) to afford monoketone 10 (2.50 g, 87%).

¹ H NMR (500MHz, CDCl ₃ ) Shift=5.73(s,1H),5.29-5.25(m,1H),3.98-3.90(m,4H),2.48(dd,J=8.8,19.5Hz,1H), 2.46-2.40(m,1H),2.36(dd,J=5.9,12.7Hz,1H),2.34-2.25(m,2H),2.24-2.08(m,5H),2.09(d,J=13.2Hz, 1H), 1.95(dd, J＝2.4, 13.2Hz, 1H), 1.90-1.81(m, 1H), 1.79-1.70(m, 2H), 1.70-1.61(m, 2H), 0.89(s, 3H) ; ¹³ C NMR (500MHz, CDCl ₃ ) Shifts = 220.9, 141.5, 140.6, 119.7, 118.6, 108.8, 81.1, 80.5, 64.7, 64.3, 47.9, 47.3, 42.5, 39.9, 36.0, 34.0, 33.9, 31.7, 28.1, 18.9, 17.0; HRMS (ESI) (m/z) C ₂₁ H ₂₇ O ₄ [M+H] ⁺ : calculated 343.1909, found 343.1919.

1-Chloroisoquinoline adduct 11

CeCl3 (565 mg, 2.30 mmol, 10.0 equiv) in a reaction flask was heated at 140 °C under vacuum for ₂ hours. Fill the flask with Ar and cool to 0 °C. After 30 minutes, THF (2.8 mL) was added and stirred at 0 °C for 2 hours. The flask was then allowed to warm to room temperature and stirred for an additional 16 hours.

To the CeCl3/THF complex solution was added ₁ -chloro-7-iodoisoquinoline (396 mg, 1.40 mmol, 6.00 equiv) in THF (1.4 mL), followed by stirring at room temperature for 10 min, then cooling to -78°C. A hexane solution of n-butyllithium (1.6M, 716 μL, 1.10 mmol, 5.00 equiv) was then added dropwise. The reaction mixture was stirred at the same temperature for an additional 30 minutes, and monoketone 10 (78.5 mg, 229 μmol) was cannulated into THF (1.4 mL). After an additional 30 minutes, saturated _NH4Cl solution (5 mL) was added to the stirred reaction mixture, which was then allowed to warm to room temperature. The mixture was diluted with EtOAc (5 mL), and the layers were separated. The aqueous layer was extracted with EtOAc (3 x 5 mL), the combined organic layers were washed with brine ( ₅ mL), dried over _Na2SO4 and concentrated under reduced pressure. The resulting residue was then purified by flash chromatography (silica gel, eluent: 2:1 hexane:EtOAc) to afford 1-chloroisoquinoline adduct 11 (115 mg, 97%).

¹ H NMR (500MHz, CDCl ₃ ) shift=8.34(br.s,1H),8.24(d,J=5.9Hz,1H),7.89-7.83(m,1H),7.76(d,J=8.3Hz, 1H), 7.56(d, J=5.9Hz, 1H), 5.63(s, 1H), 5.16-4.99(m, 1H), 4.02-3.87(m, 4H), 2.62(ddd, J=4.4, 9.8, 14.2Hz, 1H), 2.48-2.38(m, 2H), 2.36-2.26(m, 3H), 2.26-2.19(m, 1H), 2.18-2.08(m, 2H), 1.96(dd, J=2.4, 13.7Hz, 1H), 1.88(dd, J＝5.1, 17.8Hz, 1H), 1.82-1.70(m, 2H), 1.67-1.57(m, 3H), 1.49(d, J＝17.6Hz, 1H), 1.20-1.08(m,3H); HRMS(ESI)(m/z) C ₂₂ H ₂₆ NaO ₅ [M+Na] ⁺ : Calculated 393.1673, Found 393.1657.

Isoquinoline 12

A solution of 1-chloroisoquinoline adduct 11 (115 mg, 227 μmol) in dichloromethane (20 mL) was cooled to 0°C. To this solution was then added pyridine (183 μL, 2.30 mmol, 10.0 equiv) and DMAP (13.9 mg, 114 μmol, 0.50 equiv). After 5 minutes, trifluoroacetic anhydride (158 μL, 1.14 mmol, 5.00 equiv) was added dropwise and stirred for an additional 30 minutes, at which point pH 7 phosphate buffer (15 mL) was added and the reaction flask was allowed to warm to room temperature. The organic and aqueous layers were separated, and the aqueous layer was extracted with dichloromethane (2 x 15 mL). The organic layers were combined, washed with brine (25 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The resulting residue was then purified by short flash column chromatography (silica gel, eluent: 2:1 hexane:EtOAc) to give the trifluoroacetylated product which was flashed to the next step.

The trifluoroacetylated product (130 mg, 216 mmol) was azeotropically dried with benzene and dissolved in benzene (4.3 mL). AIBN (106 mg, 647 μmol, 3.00 equiv) was added and the reaction flask was degassed by cryopump thawing process (3 cycles). _Bu3SnH (1.16 mL, 4.31 mmol, 20.0 equiv) was added and the reaction mixture was warmed to reflux. After 3 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was then purified by flash column chromatography (silica gel, eluent: 4:1 to 3:1 to 1:1 hexane:EtOAc) to afford isoquinoline 12 (67.0 mg, 65% for two steps).

¹ H NMR (500MHz, CDCl ₃ ) shift=9.21(s,1H),8.46(d,J=5.9Hz,1H),7.77(s,1H),7.73(d,J=8.3Hz,1H),7.61 (d,J=5.9Hz,1H),7.57(d,J=8.3Hz,1H),5.74(s,1H),5.29-5.23(m,1H),4.00-3.90(m,4H),3.11( t,J=10.0Hz,1H),2.49(dd,J=8.3,11.2Hz,1H),2.47-2.41(m,1H),2.38-2.24(m,4H),2.24-2.14(m,2H) ,2.12(d,J=13.2Hz,1H),2.06-1.95(m,2H),1.91(dd,J=5.4,17.6Hz,1H),1.83(dq,J=4.9,11.7Hz,1H), 1.77(td,J=2.3,12.9Hz,1H),1.72-1.59(m,3H),0.52(s,3H); ¹³ C NMR(500MHz,CDCl ₃ ) shift=152.4,142.6,141.2,140.6,140.2 ,134.7,132.1,128.7,126.4,125.8,120.2,119.9,119.3,108.9,81.4,80.3,64.7,64.3,57.1,51.8,44.9,42.6,40.1,39.8,34.2,30.9,28.2,36.5,1 ; HRMS (ESI) (m/z) C ₃₀ H ₃₃ NaNO ₃ [M+Na] ⁺ : calculated 478.2353, found 478.2347.

Ketone 13

To a solution of isoquinoline 12 (365 mg, 0.801 mmol, 1.00 equiv) in acetone and water (4:1, 0.025M) was added PTSA (412 mg, 2.16 mmol, 2.70 equiv) and the reaction mixture was heated to 55 °C. After 14.5 hours, the reaction was cooled to room temperature, and saturated NaHCO ₃ solution and ethyl acetate were sequentially added to the reaction. The layers were separated and the aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure. The resulting residue was then purified by flash chromatography (silica gel, eluent: 3:2→1:2 hexane:EtOAc) to afford ketone 13 (289 mg, 87%).

¹ H NMR (500MHz, CDCl ₃ ) Shift=9.23(s,1H),8.48(d,J=5.9Hz,1H),7.80(s,1H),7.78(d,J=8.3Hz,1H),7.65 (d, J=5.9Hz, 1H), 7.61(d, J=8.3Hz, 1H), 5.91(s, 1H), 5.40-5.35(m, 1H), 3.15(t, J=10.0Hz, 1H) ,2.94(d,J=15.1Hz,1H),2.68(d,J=15.1Hz,1H),2.67-2.59(m,1H),2.58-2.41(m,4H),2.41-2.24(m,3H ),2.24-2.10(m,2H),2.04(tt,J＝4.6,13.2Hz,1H),1.96(dd,J＝5.4,17.6Hz,1H),1.86(dq,J＝5.1,12.1Hz, 1H), 1.80-1.67(m, 2H), 0.55(s, 3H); ¹³ CNMR (500MHz, CDCl ₃ ) shift = 208.9, 152.2, 142.2, 140.3, 140.2, 139.4, 134.9, 132.3, 128.7, 126.5, 126.0 ,121.5,120.9,120.4,82.8,80.4,57.1,51.7,49.2,44.8,40.1,40.0,39.8,30.8,29.8,28.1,26.5,20.7,15.4; HRMS(ESI)(m/z)C ₂₈ H ₃₀ NO ₂ [M+H] ⁺ : calculated value 412.2271, measured value 412.2288.

Scheme 1-4. Optimizing route 3

Triflate

To a solution of monoketone 10 (2.50 g, 7.30 mmol, 1.00 equiv) in THF (45 mL) was added NaHMDS (1M, 8.76 mL, 8.76 mmol, 1.20 equiv) dropwise at -78°C. After stirring for 1.5 h, _PhNTf2 (3.91 g, 11.0 mmol, 1.50 equiv) in THF (20 mL) was cannulated and the reaction mixture was warmed to 0 °C. After another 30 minutes, saturated _NH4Cl solution (50 mL) was added to the stirred reaction mixture and diluted with EtOAc (70 mL). The layers were separated, the aqueous layer was extracted with EtOAc (2 x 45 mL), the combined organic layers were washed with brine (80 mL), dried _{over Na2SO4} , and concentrated under reduced pressure. The resulting residue was then purified by flash column chromatography (silica gel, eluent: 8:1 → 5:1 hexane:EtOAc) to afford the triflate (3.33 g, Calculate the yield after coupling).

¹ H NMR (500MHz, CDCl ₃ ) shift=5.76(s,1H),5.67(br.s.,1H),5.32(dd,J=2.0,4.9Hz,1H),4.02-3.94(m,4H) ,2.67(dd,J=6.8,10.7Hz,1H),2.49(t,J=14.6Hz,1H),2.45(ddd,J=3.7,6.5,15.2Hz,1H),2.38-2.28(m,4H ), 2.17(ddd, J=1.5, 10.7, 12.7Hz, 1H), 2.12(d, J=13.2Hz, 1H), 2.10(dd, J=5.9, 17.6Hz, 1H), 1.98(dd, J= 2.7,13.4Hz,1H),1.88(ddd,J＝7.6,8.9,12.8Hz,1H),1.80(tdd,J＝2.4,4.8,12.7Hz,1H),1.74-1.63(m,2H),1.03 (s,3H); HRMS(ESI)(m/z) C ₂₂ H ₂₆ O ₆ F ₃ S[M+H] ⁺ : calculated 475.1397, found 475.1411.

C16-C17 unsaturated isoquinoline

To triflate (3.33mg, 7.02mmol, 1.00eq) and isoquinoline-7-boronic acid (3.64g, 21.1mmol, 3.00eq) in 1,4-dioxane (300mL) and H ₂ O To the solution in (30 mL) was added K2CO3 (2.91 _g , 21.1 mmol, _3.00 eq) and the solution was bubbled through inert Ar for 5 min. Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (286 mg, 350 μmol, 0.05 equiv) was added and the reaction mixture was stirred at 80° C. for 1 hour. The mixture was cooled to room temperature, and saturated NaHCO ₃ solution (200 mL) was added. The mixture was diluted with EtOAc (350 mL), and the layers were separated. The aqueous layer was extracted with EtOAc (2 x 300 mL), the combined organic layers were washed with brine (500 mL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (silica gel, eluent: 2:1 → 1:1 → 1:2 hexane:EtOAc) to provide C16-C17 unsaturated isoquinoline (2.67 mg, 84% for 2 steps) .

¹ H NMR (500MHz, CDCl ₃ ) shift = 9.23 (s, 1H), 8.49 (d, J = 5.4Hz, 1H), 7.94 (s, 1H), 7.85-7.81 (m, 1H), 7.80-7.75 ( m,1H),7.63(d,J=5.4Hz,1H),6.26(br.s.,1H),5.82(s,1H),5.40(d,J=3.4Hz,1H),4.08-3.90( m, 4H), 2.76(dd, J=7.1, 11.0Hz, 1H), 2.58(dt, J=5.4, 17.6Hz, 1H), 2.56-2.40(m, 3H), 2.40-2.28(m, 4H) ,2.16(d,J=13.2Hz,1H),2.02(dd,J=2.0,13.2Hz,1H),1.94(td,J=8.8,13.2Hz,1H),1.81(td,J=2.0,12.7 Hz, 1H), 1.76-1.67(m, 2H), 1.18(s, 3H; HRMS(ESI)(m/z) calculated value C ₃₀ H ₃₂ NO ₃ [M+H] ⁺ : 454.2377, measured value 454.2366.

Isoquinoline 12

To a solution of 17,18-unsaturated isoquinoline (534 mg, 1.17 mmol, 1.00 eq) in THF (48 mL) was added 10 wt% Pd/C (374 mg, 351 μmol, 0.30 eq) with a _H2 balloon installed. After 3 hours, the reaction mixture was filtered through a pad of celite, washed with 0.2M _NH3 in MeOH (50 mL), and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, eluent: 40:1→30:1 in DCM:MeOH) to afford isoquinoline 12 (452 mg, 84%).

Example S2. Synthesis of lactams of formula (A-1), (A-1') or (A-1") and (A-2') or (A-2")

lactam 15B

To a solution of ketone 13 (5.5 mg, 13.6 μmol, 1.00 equiv) in MeOH (350 μL) was added H2NOH _· HCl (2.5 mg, 27.2 μmol, 2.00 equiv) and NaOAc (4.9 mg, 27.2 μmol, 2.00 equiv). After stirring at 70°C for 1.5 hours, the reaction mixture was cooled to room temperature and roughly concentrated. H ₂ O (300 μL) was added, extracted with ethyl acetate (3×300 μL), the combined organic phases were washed with brine (300 μL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The crude mixture was used in the next step without further purification.

To a solution of the crude mixture (13.6 μmol, 1.00 equiv) in DCM (350 μL) was added trimethylamine (11.4 μL, 81.6 μmol, 6.00 equiv). At 0 °C, methanesulfonic anhydride (4.7 mg, 27.2 μmol, 2.00 equiv) was added. The reaction mixture was stirred at 0°C for 15 minutes, warmed to room temperature and stirred for an additional 15 minutes. The reaction mixture was quenched with NaHCO ₃ (300 μL) and extracted with DCM (3×300 μL), the combined organic phases were washed with brine (300 μL), dried over Na ₂ SO ₄ and concentrated under reduced pressure. The crude mixture was used in the next step without further purification.

The crude mixture was dissolved in AcOH (300 μL) and stirred at 50° C. for 16 hours. The reaction mixture was roughly concentrated and NaHCO ₃ (300 μL) was applied. Extracted with ethyl acetate (3 x 300 μL), the combined organic phases were washed with brine (300 μL), dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The crude mixture was purified by preparative TLC (silica gel, eluent: 5:5:1 EtOAc:DCM:TEA) to afford lactam 15B (1.5 mg, 26% for three steps).

¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.49(d,J=5.9Hz,1H),7.79(s,1H),7.76(d,J=8.2Hz,1H),7.63 (d, J=5.3Hz, 1H), 7.58(dd, J=1.5, 8.5Hz, 1H), 5.87(s, 1H), 5.78(t, J=6.5Hz, 1H), 5.34(dd, J= 2.6,5.0Hz,1H),3.57(dd,J=5.6,15.0Hz,1H),3.33(dd,J=7.6,15.3Hz,1H),3.15(dd,J=9.1,10.9Hz,1H), 2.66(ddd,J＝4.7,10.0,14.7Hz,1H),2.62-2.53(m,2H),2.52-2.46(m,2H),2.35(br.s.,1H),2.38-2.30(m, 1H),2.28-2.22(m,1H),2.22-2.12(m,2H),2.01(qt,J＝4.1,9.4Hz,1H),1.96(dd,J＝5.3,17.6Hz,1H),1.90 -1.79(m, J＝5.3, 12.3, 12.3Hz, 1H), 1.75(td, J＝8.2, 12.3Hz, 1H), 1.68(dt, J＝7.3, 10.7Hz, 1H), 0.54(s, 3H ).HRMS(ESI)(m/z) C ₂₈ H ₃₁ N ₂ O ₂ [M+H] ⁺ : Calculated 427.2380, Found 427.2395.

Example S3. Reductive amination

Method A

To a solution of ketone 13 (1.00 equiv) in 1,2-dichloroethane (0.02M) was added sequentially amine (4.00 equiv), AcOH (1.50 equiv) and _NaBH3CN (3.50 equiv) at room temperature. If the reactive amine was in the form of the HCl salt, triethylamine (4 equiv) was added (Method AA). Once the reaction was complete, saturated NaHCO ₃ solution was added and the layers were separated. The aqueous layer was extracted with dichloromethane. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure. (α-NR ₂ : β-NR ₂ = ~1:1.2 to ~1:5).

Method B

To a solution of ketone 13 (1.00 equiv) in 1,2-dichloroethane (0.02M) was added sequentially amine (2.00 equiv), AcOH (2.00 equiv) and NaBH(OAc) ₃ (2.00 equiv) at room temperature. If the reactive amine was in the form of the HCl salt, triethylamine (2.00 equiv) was added (Method BB). Once the reaction was complete, saturated NaHCO ₃ solution was added and the layers were separated. The aqueous layer was extracted with dichloromethane. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure. (α-NR ₂ : β-NR ₂ = ~1:1.2 to ~1:5).

Method C

To a solution of the secondary amine (1.00 equiv) in dichloromethane (0.02M) was added formaldehyde or acetaldehyde (5.00 equiv) and stirred at room temperature for 1 hour, then NaBH(OAc) ₃ (2.00 equiv) was added. Once the reaction was complete, saturated NaHCO ₃ solution was added and the layers were separated. The aqueous layer was extracted with dichloromethane. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure.

Method D: General method in favor of α-amines

To a solution of ketone 13 (1.00 equiv) in THF and t-BuOH (4:1, 0.02M), add amine (5.00 equiv) and Ti(Oi-Pr) ₄ (3.00 equiv) successively, and stir at room temperature for 15 hours ( ₄ hours for Me2NH, _MeNH2 and _NH3 ). The reaction mixture was cooled to -20 °C and NaBH4 ₍ 1.50 equiv) was added. Once the reaction was complete, saturated NaHCO ₃ solution was added and the layers were separated. The aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure. (α-NR ₂ :β-NR ₂ = ~1.1:1 to ~3.7:1).

Method E: General Procedure for Methanesulfonamide Formation

To a solution of the amine (1.00 equiv) in dichloromethane (0.013M) was added trimethylamine (4.00 equiv) and the reaction mixture was cooled to -20°C. Methanesulfonic anhydride (2.50 equivalents) was added as a dichloromethane solution, and stirred at the same temperature for 30 minutes. 2N NaOH solution was added and the layers were separated. The aqueous layer was extracted with dichloromethane. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure.

β-dimethylamine 14B and α-dimethylamine 14A

The crude mixture was sequentially purified by flash chromatography (silica gel, eluent: 20:1 EtOAc:2M _NH3 in MeOH) to afford β-dimethylamine 14B (21.5 mg, 65%). Ca. 0.6 _mg α- Dimethylamine 14A.

β-Dimethylamine 14B: ¹ H NMR (500MHz, C ₆ D ₆ ) Shift=9.31(s,1H),8.61(d,J=5.4Hz,1H),7.43(s,1H),7.39(d, J＝8.8Hz, 1H), 7.25(d, J＝5.4Hz, 1H), 7.23(d, J＝8.8Hz, 1H), 5.73(br.s, 1H), 5.18(s, 1H), 2.74( t,J=10.0Hz,1H),2.63(dd,J=8.8,11.2Hz,1H),2.48-2.28(m,2H),2.27-2.20(m,1H),2.19-2.03(m,6H) ,2.00(br.s,6H),1.95-1.84(m,2H),1.83-1.66(m,5H),1.41(tt,J=5.4,13.2Hz,1H),0.45(s,3H).HRMS (ESI)(m/z)C ₃₀ H ₃₇ N ₂ O[M+H] ⁺ : calculated value 441.2900, found value 441.2910.

α-Dimethylamine 14A: ¹ H NMR (600MHz, C ₆ D ₆ ) Shift = 9.26(s, 1H), 8.56(d, J=5.9Hz, 1H), 7.44-7.39(m, 1H), 7.36( d,J=8.2Hz,1H),7.21-7.20(m,1H),7.20(d,J=5.9Hz,1H),5.68-5.65(m,1H),5.15-5.11(m,1H),2.72 -2.66(m, J=10.0Hz, 1H), 2.59(dd, J=8.8, 11.2Hz, 1H), 2.34(tt, J=2.9, 12.1Hz, 1H), 2.16(td, J=3.2, 16.0 Hz,1H),2.09(s,6H),2.13-1.92(m,8H),1.85(ddd,J=5.0,9.0,13.6Hz,1H),1.73(dt,J=5.3,12.3Hz,1H) ,1.72-1.66(m,2H),1.60-1.57(m,1H),1.57-1.49(m,1H),1.20(dq,J＝4.1,12.3Hz,1H),0.40(s,3H).HRMS (ESI)(m/z)C ₃₀ H ₃₇ N ₂ O[M+H] ⁺ : calculated value 441.2900, found value 441.2909.

β-morpholine 15B and α-morpholine 15A

β-morpholine 15B: Purification of the crude mixture by flash chromatography (silica gel, eluent: 100% EtOAc→35:1→20:1→10:1 EtOAc:MeOH) afforded β-morpholine 15B (21 mg, 66%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.3Hz,1H),7.62 (d,J=5.9Hz,1H),7.59(dd,J=1.0,8.8Hz,1H),5.71(s,1H),5.24(d,J=2.9Hz,1H),3.73(br.s, 4H), 3.13(t, J＝10.0Hz, 1H), 2.65-2.28(m, 11H), 2.23-2.11(m, 3H), 2.06(d, J＝13.2Hz, 1H), 2.01(dt, J =4.4,9.0Hz,1H),1.93(dd,J=4.9,17.1Hz,1H),1.89-1.79(m,1H),1.75-1.53(m,4H),0.54(s,3H).HRMS( ESI) (m/z) C ₃₂ H ₃₉ N ₂ O ₂ [M+H] ⁺ : calculated value 483.3006, found value 483.3012.

β-N-methylpiperazine 16B and α-N-methylpiperazine 16A

β-N-methylpiperazine 16B: by flash chromatography (silica gel, first column: eluent: 100% MeOH → 10: ₁ EtOAc: 2M NH in MeOH / second column: eluent: 20:1 EtOAc:2M _NH3 in MeOH)) sequential purification of the crude mixture afforded β-N-methylpiperazine 16B (20 mg, 55%). ¹ H NMR (600MHz, CDCl ₃ ) shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H),7.62 (d,J=5.9Hz,1H),7.59(d,J=8.2Hz,1H),5.70(s,1H),5.25-5.22(m,1H),3.13(t,J=9.7Hz,1H) ,2.53(br.s.,1H),2.50(dd,J=8.8,11.7Hz,1H),2.41(t,J=12.9Hz,1H),2.38-2.33(m,3H),2.32(br. s,3H),2.22-2.11(m,3H),2.10-1.95(m,3H),1.95-1.89(m,2H),1.84(dq,J＝5.3,11.7Hz,1H),1.79-1.50( m,11H),0.62-0.43(m,3H).HRMS(ESI)(m/z)C ₃₃ H ₄₂ N ₃ O[M+H] ⁺ : calculated value 496.3322, measured value 496.3337.

β-azetidine 18B and α-azetidine 18A

β-azetidine 18B: The crude mixture was purified by preparative TLC (eluent: 1:1 EtOAc:MeOH) to afford β-azetidine 18B (2.7 mg, 50%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H),7.62 (d,J=5.4Hz,1H),7.59(d,J=8.3Hz,1H),5.69(s,1H),5.22(d,J=2.4Hz,1H),3.20-3.05(m,5H) ,2.59-2.43(m,4H),2.39-2.28(m,2H),2.23-2.12(m,2H),2.07-1.96(m,4H),1.92(dd,J＝5.1,17.3Hz,1H) ,1.89-1.79(m,3H),1.75-1.55(m,3H),1.40(t,J=13.2Hz,1H),0.54(s,3H).HRMS(ESI)(m/z)C ₃₁ H ₃₇ N ₂ O[M+H] ⁺ : 4 calculated value 53.2906, measured value 453.2916.

β-Pyrrolidine 19B and α-Pyrrolidine 19A

β-pyrrolidine 19B: The crude mixture was purified by preparative TLC (eluent: 20:10: ₃ EtOAc:hexane:2M NH in MeOH) to afford β-pyrrolidine 19B (2.0 mg, 40%) . ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.48(d,J=5.4Hz,1H),7.79(s,1H),7.75(d,J=8.3Hz,1H),7.62 (d,J=5.9Hz,1H),7.59(d,J=8.3Hz,1H),5.70(br.s.,1H),5.22(br.s.,1H),3.13(t,J=9.8 Hz,1H),2.59-2.46(m,6H),2.44(br.s.,1H),2.41-2.28(m,3H),2.23-2.12(m,2H),2.11-2.00(m,2H) ,2.00-1.82(m,4H),1.79-1.65(m,6H),1.64-1.51(m,2H),0.54(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₃₉ N ₂ O[M+H] ⁺ : calculated value 467.3057, measured value 467.3053.

β-dimethylamine 17,18-unsaturated isoquinoline 23B and α-dimethylamine 17,18-unsaturated isoquinoline 23A

β-Dimethylamine 17,18-unsaturated isoquinoline 23B: Sequential purification of the crude mixture by flash chromatography (silica gel, eluent: 20:1 EtOAc:2M _NH3 in MeOH) afforded β-dimethylamine Amine 17,18-unsaturated isoquinoline 23B (6.5 mg, 74%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.24(br.s.,1H),8.51(d,J=5.4Hz,1H),7.94(s,1H),7.84-7.76(m,2H),7.63 (d,J=5.4Hz,1H),6.27(br.s.,1H),5.97(s,1H),5.50(dd,J=2.4,4.9Hz,1H),2.98(d,J=14.6Hz ,1H),2.78(dd,J=6.8,11.2Hz,1H),2.71(d,J=14.6Hz,1H),2.72-2.63(m,1H),2.61(d,J=5.4Hz,1H) ,2.59-2.54(m,2H),2.54-2.50(m,2H),2.50-2.42(m,2H),2.39(ddd,J＝1.5,11.0,12.9Hz,1H),2.20(ddd,J＝ 1.5,9.5,11.5Hz,1H),2.01(ddd,J＝7.3,8.8,12.7Hz,1H),1.79(dt,J＝7.3,11.2Hz,1H),1.18(s,3H).HRMS(ESI )(m/z)C ₃₀ H ₃₅ N ₂ O[M+H] ⁺ : calculated value 439.2744, found value 439.2753.

β-methylamine 24B and α-methylamine 24A

β-monomethylamine 24B: The crude mixture was purified by preparative TLC (eluent: 10:1 EtOAc:2M _NH3 in MeOH) to afford β-monomethylamine 24B (ca. 1.5 mg, 58% ). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.48(d,J=5.4Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H),7.62 (d,J=5.9Hz,1H),7.59(dd,J=1.0,8.3Hz,1H),5.72(d,J=1.0Hz,1H),5.24(dd,J=2.2,5.1Hz,1H) ,3.13(t,J=10.0Hz,1H),3.03-2.98(m,1H),2.57-2.50(m,1H),2.51(dd,J=8.3,11.7Hz,1H),2.44(s,3H ), 2.36(d, J=15.2Hz, 1H), 2.36-2.28(m, 2H), 2.26-2.13(m, 2H), 2.09(dd, J=3.7, 16.4Hz, 1H), 2.07-1.99( m,2H),1.98-1.92(m,1H),1.93(dd,J=5.9,17.6Hz,1H),1.85(dq,J=4.9,11.7Hz,1H),1.82-1.76(m,1H) ,1.76-1.58(m,3H),0.54(s,3H).HRMS(ESI)(m/z)C ₂₉ H ₃₅ N ₂ O[M+H] ⁺ : calculated value 427.2744, found value 427.2740.

β-Deuterated dimethylamine 26B and α-Deuterated dimethylamine 26A

β-Deuterated dimethylamine 26B: Add triethylamine. The crude mixture was sequentially purified by flash chromatography (silica gel, eluent: 20:1 EtOAc:2M _NH3 in MeOH) to afford β-deuterodimethylamine 26B (4 mg, 62%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.80(s,1H),7.77(d,J=8.3Hz,1H),7.64 (d,J=5.9Hz,1H),7.60(d,J=8.3Hz,1H),5.74(br.s.,1H),5.26(br.s.,1H),3.15(t,J=10.0 Hz, 1H), 2.51(dd, J=8.8, 11.2Hz, 1H), 2.50-2.42(m, 1H), 2.37(d, J=17.1Hz, 1H), 2.38-2.26(m, 2H), 2.26 -2.09(m,4H),2.08-1.98(m,2H),1.95(dd,J＝5.1,17.3Hz,1H),1.87(dq,J＝5.4,12.2Hz,1H),1.80-1.68(m ,3H),1.62(br.s.,2H),0.63-0.50(s,3H).HRMS(ESI)(m/z)C ₃₀ H ₃₁ D ₆ N ₂ O[M+H] ⁺ : calculated value 447.3277, measured value 447.3281.

β-2-methoxyethylmethylamine 27B and α-2-methoxyethylmethylamine 27A

β-2-Methoxyethylmethylamine 27B: Purification of the crude mixture by preparative TLC (eluent: 10:10:1 Hexane:EtOAc:2M NH in MeOH) gave β- ₂ -methanol Oxyethylmethylamine 27B (ca. 1.2 mg, 20%). ¹ H NMR (500MHz, CD ₃ OD) shift=9.21(s,1H),8.40(d,J=5.4Hz,1H),7.99(s,1H),7.90(d,J=8.8Hz,1H), 7.81(d, J=5.9Hz, 1H), 7.77(s, 1H), 5.80(s, 1H), 5.35-5.27(m, 1H), 3.60(t, J=5.4Hz, 2H), 3.39(s ,2H),3.24(t,J=10.0Hz,1H),3.15-2.88(m,2H),2.56(br.s.,3H),2.51(dd,J=9.3,10.7Hz,1H),2.48 -2.39(m,3H),2.35-2.28(m,1H),2.25-2.09(m,4H),2.02-1.84(m,7H),1.76(s,2H),0.59(s,3H).HRMS (ESI)(m/z) C ₃₂ H ₄₁ N ₂ O ₂ [M+H] ⁺ : Calculated 485.3163, Found 485.3170.

β-bis-2-methoxyethylamine 28B and α-bis-2-methoxyethylamine 28A

β-bis-2-methoxyethylamine 28B: The crude mixture was purified by preparative TLC (eluent: 10:1 dichloromethane:MeOH) to afford β-bis-2-methoxyethylamine 28B ( ca. 1.1 mg, 19%). ¹ H NMR (500MHz, CD ₃ OD) shift=9.21(s,1H),8.39(d,J=5.9Hz,1H),7.99(s,1H),7.90(d,J=8.8Hz,1H), 7.81(d, J=5.9Hz, 1H), 7.77(s, 1H), 5.74(s, 1H), 5.29-5.24(m, 1H), 3.47(t, J=6.1Hz, 4H), 3.36(s ,6H),3.24(t,J=10.5Hz,1H),3.06-2.93(m,1H),2.78(d,J=5.9Hz,4H),2.52(dd,J=9.0,11.5Hz,1H) ,2.49-2.43(m,1H),2.44(d,J＝17.6Hz,1H),2.40-2.35(m,1H),2.35-2.26(m,1H),2.24-2.10(m,3H),2.07 -1.94(m,3H),1.91(dd,J=5.4,17.6Hz,1H),1.85(d,J=14.6Hz,1H),1.82-1.67(m,3H),0.59(s,3H). HRMS(ESI)(m/z) C ₃₄ H ₄₅ N ₂ O ₃ [M+H] ⁺ : Calculated 529.3425, Found 529.3434.

β-2-fluoroethylmethylamine 29B and α-2-fluoroethylmethylamine 29A

β-2-fluoroethylmethylamine 29B: The crude mixture was purified by prep-TLC (eluent: 20:1 dichloromethane:MeOH) to afford β-2-fluoroethylmethylamine 29B (2.7 mg, 51 %). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.80(s,1H),7.77(d,J=8.8Hz,1H),7.64 (d,J=5.9Hz,1H),7.60(dd,J=1.0,8.3Hz,1H),5.74(br.s.,1H),5.26(br.s.,1H),4.68-4.46(m ,2H),3.15(t,J=9.8Hz,1H),2.99-2.69(m,3H),2.52(dd,J=8.8,11.2Hz,1H),2.47-2.30(m,6H),2.29- 2.16(m,4H),2.16-2.00(m,3H),2.01-1.92(m,1H),1.94(dd,J=5.1,17.3Hz,1H),1.86(dq,J=5.4,12.2Hz, 1H),1.79-1.64(m,3H),0.56(s,3H).HRMS(ESI)(m/z)C ₃₁ H ₃₈ N ₂ OF[M+H] ⁺ : calculated value 473.2963, found value 473.2971.

β-2,2-difluoroethylmethylamine 30B and α-2,2-difluoroethylmethylamine 30A

β-2,2-difluoroethylmethylamine 30B: The crude mixture was purified by prep-TLC (eluent: 1:1 hexane:EtOAc) to afford β-2,2-difluoroethylmethylamine 30B (ca. 1.1 mg, 19%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.28(s,1H),8.50(d,J=5.9Hz,1H),7.86(s,1H),7.83(d,J=8.3Hz,1H),7.75 (d,J=5.4Hz,1H),7.69(d,J=8.3Hz,1H),6.15-5.83(m,1H),5.75(s,1H),5.27(dd,J=2.4,5.4Hz, 1H), 3.17(t, J=10.0Hz, 1H), 2.91(br.s., 2H), 2.52(dd, J=8.8, 11.7Hz, 1H), 2.47(br.s, 3H), 2.45- 2.30(m,4H),2.27-2.11(m,6H),2.11-1.99(m,2H),1.95(dd,J＝5.1,17.3Hz,1H),1.88(dq,J＝6.3,12.7Hz, 1H),1.77-1.68(m,3H),0.56(s,3H).HRMS(ESI)(m/z)C ₃₁ H ₃₇ N ₂ OF ₂ [M+H] ⁺ : calculated value 491.2868, measured value 491.2879 .

β-7-Azabicyclo[2.2.1]heptane 31B and α-7-azabicyclo[2.2.1]heptane 31A

β-7-Azabicyclo[2.2.1]heptane 31B: by flash chromatography (silica gel, eluent: 10:10:1 → 10:10:2 in hexane:EtOAc:2M _NH3 in MeOH ) purified the crude mixture to afford β-7-azabicyclo[2.2.1]heptane 31B (3 mg, 50%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.81(s,1H),7.77(d,J=8.3Hz,1H),7.64 (d,J=5.9Hz,1H),7.61(d,J=8.3Hz,1H),5.71(br.s.,1H),5.24(br.s.,1H),3.43(br.s., 2H), 3.15(t, J=9.8Hz, 1H), 2.69(br.s., 2H), 2.52(t, J=9.8Hz, 1H), 2.46(br.s., 1H), 2.37(d ,J=17.6Hz,1H),2.38-2.29(m,1H),2.27-2.13(m,3H),2.11-1.92(m,6H),1.87(dq,J=5.9,12.7Hz,1H), 1.86-1.79(m,1H),1.78-1.60(m,8H),1.55(t,J=13.2Hz,1H),0.56(s,3H).HRMS(ESI)(m/z)C ₃₄ H ₄₁ N ₂ O[M+H] ⁺ : calculated value 493.3213, measured value 493.3224.

β-isopropylamine 32B and α-isopropylamine 32A

β-Isopropylamine 32B: The crude mixture was purified by flash chromatography (silica gel, eluent: 10:1 EtOAc:2M _NH3 in MeOH) to afford β-isopropylamine 32B (5 mg, 70%). ¹ H NMR (600MHz, CDCl ₃ ) shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H),7.62 (d,J=5.9Hz,1H),7.59(d,J=8.2Hz,1H),5.71(s,1H),5.24(d,J=2.9Hz,1H),3.24(br.s.,1H ), 3.13(t, J=9.7Hz, 1H), 2.90(br.s., 1H), 2.50(dd, J=8.5, 11.4Hz, 2H), 2.41-2.26(m, 3H), 2.24-2.13 (m,3H),2.09(dd,J=2.9,15.3Hz,1H),2.06-1.98(m,2H),1.93(dd,J=5.3,17.6Hz,1H),1.85(dq,J=5.3 ,12.3Hz,1H),1.75-1.62(m,4H),1.07(br.s.,6H),0.54(s,3H).HRMS(ESI)(m/z)C ₃₁ H ₃₉ N ₂ O[ M+H] ⁺ : calculated value 455.3057, measured value 493.3049.

β-isopropylmethylamine 33B

The crude mixture was purified by preparative TLC (eluent: 20:10:3 hexane:EtOAc:2M _NH3 in MeOH) to afford β-isopropylmethylamine 33B (4 mg, 78%). ¹ H NMR (600MHz, CDCl ₃ ) shift=9.22(br.s.,1H),8.49(d,J=4.7Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H ), 7.62(d, J=5.3Hz, 1H), 7.59(d, J=8.2Hz, 1H), 5.70(br.s., 1H), 5.22(br.s., 1H), 3.22(br. s.,1H),3.13(t,J=10.0Hz,1H),2.77(br.s.,1H),2.50(t,J=8.2Hz,1H),2.43(t,J=12.9Hz,1H ), 2.36(d, J=17.6Hz, 1H), 2.35-2.28(m, 2H), 2.22-2.14(m, 2H), 2.17(dt, J=4.4, 9.0Hz, 1H), 2.11(br. s.,3H),2.08-1.99(m,2H),1.98-1.91(m,1H),1.93(dd,J＝4.1,17.0Hz,1H),1.85(dq,J＝5.3,12.3Hz,1H ),1.69(br.s.,2H),1.59(br.s.,1H),1.56(br.s.,1H),0.96(br.s.,6H),0.54(s,3H).HRMS (ESI)(m/z)C ₃₂ H ₄₁ N ₂ O[M+H] ⁺ : Calculated 455.3057, Found 493.3049.

β-isopropylethylamine 34B

The crude mixture was purified by preparative TLC (eluent: 100% MeOH) to afford β-isopropylethylamine 34B (ca. 1.5 mg, 20%). ¹ H NMR (600MHz, CD ₃ OD) shift=9.19(s,1H),8.38(d,J=5.9Hz,1H),7.97(s,1H),7.88(d,J=8.8Hz,1H), 7.79(d, J=5.9Hz, 1H), 7.74(d, J=8.2Hz, 1H), 5.76(br.s., 1H), 5.27(br.s., 1H), 3.26-3.19(m, 1H), 2.91-2.66(m, 3H), 2.50(dd, J＝9.4, 11.2Hz, 1H), 2.48-2.36(m, 3H), 2.29(t, J＝10.6Hz, 1H), 2.23-2.06 (m,4H),2.00-1.86(m,5H),1.86-1.79(m,1H),1.79-1.66(m,2H),1.21-1.08(m,9H),0.57(s,3H).HRMS (ESI)(m/z)C ₃₃ H ₄₃ N ₂ O[M+H] ⁺ : Calculated 483.3370, Found 483.3382.

β-(R)-3-fluoropyrrolidine 35B and α-(R)-3-fluoropyrrolidine 35A

β-(R)-3-fluoropyrrolidine 35B: The crude mixture was purified by preparative TLC (eluent: 47.5:47.5: ₅ EtOAc:hexane:2M NH in MeOH) to give β-(R)- 3-Fluoropyrrolidine 35B (2.2 mg, 38%). ¹ H NMR (500MHz, CDCl ₃ ) shift=δ9.22(s,1H),8.49(d,J=5.4Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H) ,7.62(d,J=5.4Hz,1H),7.59(d,J=8.8Hz,1H),5.71(d,J=2.0Hz,1H),5.23(m,1H),5.11(m,1H) ,3.40(m,1H),3.13(dd,J＝9.3,9.3Hz,1H),2.88-2.96(m,2H),2.66-2.77(m,1H),2.48-2.58(m,3H),2.29 -2.45(m,3H),2.12-2.23(m,3H),1.99-2.09(m,5H),1.83-1.97(m,2H),1.67-1.74(m,2H),1.59(m,1H) ,0.55(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₃₈ FN ₂ O[M+H] ⁺ : Calculated 485.2968, Found 485.2915.

β-(S)-3-fluoropyrrolidine 36B and α-(S)-3-fluoropyrrolidine 36A

β-(S)-3-fluoropyrrolidine 36B: The crude mixture was purified by preparative TLC (eluent: 47.5:47.5: ₅ EtOAc:hexane:2M NH in MeOH) to give β-(S)- 3-Fluoropyrrolidine 36B (2.7 mg, 46%). ¹ H NMR (500MHz, CD ₃ OD) Shift=9.21-9.18(m,1H),8.37(d,J=5.87Hz,1H),7.98-7.96(m,1H),7.88(d,J=8.80Hz ,1H),7.80-7.77(m,1H),7.74(dd,J=8.56,1.71Hz,1H),5.71(d,J=1.47Hz,1H),5.23(m,1H),5.22-5.07( m,2H),3.22(t,J=9.8Hz,1H),3.08-2.97(m,1H),2.90(td,J=8.19,5.62Hz,1H),2.70-2.64(m,1H),2.62 -2.58(m,1H),2.52-2.34(m,6H),2.33-2.24(m,1H),2.23-2.03(m,3H),2.03-1.85(m,6H),1.77-1.67(m, 2H),1.67-1.56(m,1H),0.57(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₃₈ FN ₂ O[M+H] ⁺ : calculated value 485.6553, measured value 485.6551.

β-3,3-difluoropyrrolidine 37B and α-3,3-difluoropyrrolidine 37A

β-3,3-Difluoropyrrolidine 37B: The crude mixture was purified by preparative TLC (eluent: 80:15:5 EtOAc:hexane:2M _NH3 in MeOH) to give β-3,3- Difluoropyrrolidine 37B (2.9 mg, 40%). ¹ H NMR (500MHz, CDCl ₃ ) shift=δ9.22(s,1H),8.49(d,J=5.4Hz,1H),7.79(s,1H),7.75(d,J=8.3Hz,1H) ,7.62(d,J=5.4Hz,1H),7.59(d,J=8.8Hz,1H),5.72(d,J=2.0Hz,1H),5.25(dd,J=5.4Hz,2.0Hz,1H ), 3.13(dd, J＝9.3Hz, 9.3Hz, 1H), 2.86-3.03(m, 2H), 2.14(dd, J＝6.9, 6.9Hz, 2H), 2.58(m, 1H), 2.50(dd ,J＝11.7,8.3Hz,2H),2.13-2.44(m,6H),1.98-2.10(m,2H),1.90-1.96(m,1H),1.83-1.89(m,2H),1.66-1.74 (m,2H),1.53-1.61(m,1H),0.55(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₃₇ F ₂ N ₂ O[M+H] ⁺ : calculated value 503.2874 The measured value is 503.2814.

α-3,3-Difluoropyrrolidine 37A: The crude mixture was purified by preparative TLC (eluent: 80:15: ₅ EtOAc:hexane:2M NH in MeOH) to give α-3,3- Difluoropyrrolidine 37A (2.9 mg, from 6.0 mg, 40%). ¹ H NMR (500MHz, CDCl ₃ ) shift=δ9.22(s,1H),8.49(d,J=5.4Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H) ,7.62(d,J=5.4Hz,1H),7.59(d,J=8.8Hz,1H),5.74(s,1H),5.28(d,J=2.4Hz,1H),3.15(dd,J= 9.3Hz, 9.3Hz, 1H), 3.01(dt, J=13.7, 2.4Hz, 2H), 2.83(dd, J=6.8, 6.8Hz, 2H), 2.52(dd, J=11.2, 8.3Hz, 2H) ,2.15-2.40(m,6H),2.02-2.07(m,3H),1.79-1.97(m,3H),1.72(m,3H),1.61(m,3H),0.54(s,3H).HRMS (ESI)(m/z) C ₃₂ H ₃₇ F ₂ N ₂ O[M+H] ⁺ : Calculated 503.2874 Found 503.2807.

β-2-oxa-6-azaspiro[3.4]octane 38B and α-2-oxa-6-azaspiro[3.4]octane 38A

β-2-oxa-6-azaspiro[3.4]octane 38B: The crude mixture was purified by preparative TLC (eluent: 47.5:47.5: ₅ EtOAc:hexane:2M NH in MeOH), β-2-Oxa-6-azaspiro[3.4]octane 38B (3.4 mg, 55%) was obtained. ¹ H NMR (500MHz, CD ₃ OD) shift=9.21(s,1H),8.39(d,J=5.4Hz,1H),7.99(s,1H),7.90(d,J=8.8Hz,1H), 7.81(d,J=5.9Hz,1H),7.76(dd,J=1.7,8.5Hz,1H),5.73-5.70(m,1H),5.28-5.23(m,1H),4.63(d,J= 2.4Hz, 4H), 3.28-3.21(m, 1H), 2.90(dd, J＝9.3, 49.8Hz, 2H), 2.62(t, J＝7.3Hz, 2H), 2.54-2.40(m, 5H), 2.36-2.27(m,2H),2.23-2.16(m,1H),2.13(s,2H),2.10-2.05(m,1H),2.02-1.88(m,6H),1.81-1.66(m,2H ),1.66-1.57(m,1H),0.58(s,3H).HRMS(ESI)(m/z)C ₃₄ H ₄₁ N ₂ O ₂ [M+H] ⁺ : calculated value 509.7015, found value 509.7013.

β-cyclopropylamine 39B and α-cyclopropylamine 39A

β-cyclopropylamine 39B: The crude mixture was purified by prep-TLC (eluent: 47.5:47.5: ₅ EtOAc:hexane:2M NH in MeOH) to give β-cyclopropylamine 39B (3.7 mg, 55%) . ¹ H NMR (500MHz, CD ₃ OD) shift=9.21(s,1H),8.39(d,J=5.9Hz,1H),7.99(s,1H),7.90(d,J=8.8Hz,1H), 7.81(d, J=5.4Hz, 1H), 7.76(dd, J=1.7, 8.5Hz, 1H), 5.73(d, J=2.0Hz, 1H), 5.28-5.24(m, 1H), 3.24(t ,J＝20.0Hz,1H),3.18-3.12(m,1H),2.54-2.40(m,4H),2.36-2.27(m,2H),2.18(s,3H),2.05-2.00(m,2H ),2.00-1.95(m,2H),1.94-1.91(m,1H),1.91-1.84(m,2H),1.77-1.66(m,3H),0.58(s,3H),0.51(d,J ＝4.4Hz, 2H), 0.39(dd, J＝2.2, 3.7Hz, 2H).HRMS(ESI)(m/z)C ₃₁ H ₃₇ N ₂ O[M+H] ⁺ : calculated value 453.6383, measured value 453.6381.

β-Cyclopropylmethylamine 40B

The crude mixture was purified by preparative TLC (eluent: 47.5:47.5:5 EtOAc:hexanes:2M _NH3 in MeOH) to afford β-methylcyclopropylamine 40B (1.1 mg, 85%). ¹ H NMR (500MHz, CD ₃ OD) Shift = 9.21(s, 1H), 8.39(d, J=5.4Hz, 1H), 8.00-7.98(m, 1H), 7.90(d, J=7.8Hz, 1H ), 7.81(d, J=5.9Hz, 1H), 7.76(dd, J=1.5, 9.3Hz, 1H), 5.78-5.74(m, 1H), 5.29-5.25(m, 1H), 3.26-3.24( m,1H),3.23(t,J=9.8Hz,1H),3.27-3.21(m,1H),2.88(t,J=1.0Hz,1H),2.56-2.49(m,1H),2.48-2.41 (m,2H),2.37(s,3H),2.33-2.26(m,1H),2.23-2.10(m,3H),2.03(d,J＝7.3Hz,2H),2.01-1.96(m,1H ),1.96-1.88(m,2H),1.88-1.82(m,1H),1.79-1.68(m,2H),0.59(m,5H),0.50-0.46(m,2H).HRMS(ESI)( m/z) C ₃₂ H ₃₉ N ₂ O[M+H] ⁺ : calculated value 467.6649, found value 467.6645.

β-3-methyl-3-oxetanamine 41B and α-3-methyl-3-oxetanamine 41A

β-3-Methyl-3-oxetanamine 41B: The crude mixture was purified by preparative TLC (eluent: 47.5:47.5: ₅ EtOAc:hexane:2M NH in MeOH) to give β- 3-Methyl-3-oxetanamine 41B (4.7 mg, 81%). ¹ HNMR (500MHz, CD ₃ OD) shift=9.21(s,1H),8.39(d,J=5.4Hz,1H),7.99(s,1H),7.90(d,J=8.8Hz,1H),7.81 (d,J=5.9Hz,1H),7.78-7.74(m,1H),5.75(d,J=1.5Hz,1H),5.29-5.26(m,1H),4.61(dd,J=3.4,5.9 Hz,2H),4.36(dd,J=5.9,8.3Hz,2H),3.27-3.21(m,1H),3.19-3.11(m,1H),2.50(d,J=8.3Hz,4H),2.29 (s,2H),2.23-2.13(m,2H),2.02-1.95(m,2H),1.94-1.87(m,3H),1.80-1.67(m,4H),1.55(br.s.,4H ), 0.59(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₃₉ N ₂ O ₂ [M+H] ⁺ : calculated 483.6643, found 483.6640.

β-N-methyl-1-(3-methyl-3-oxetanyl)methanamine 42B and α-N-methyl-1-(3-methyl-3-oxetane base) methylamine 42A

β-N-methyl-1-(3-methyl-3-oxetanyl)methanamine 42B: by preparative TLC (eluent: 47.5:47.5:5 EtOAc:hexane:2M NH ₃ in MeOH) to afford β-N-methyl-1-(3-methyl-3-oxetanyl)methanamine 42B (1.5 mg, 24%). ¹ H NMR (500MHz, CD ₃ OD) shift=9.22(s,1H),8.39(d,J=5.9Hz,1H),7.99(s,1H),7.90(d,J=8.3Hz,1H), 7.81(d,J=5.9Hz,1H),7.76(dd,J=1.7,8.5Hz,1H),5.75-5.72(m,1H),5.31-5.27(m,1H),4.57-4.51(m, 2H), 4.32(dd, J=1.5, 5.9Hz, 2H), 3.28-3.22(m, 1H), 2.69(s, 2H), 2.57-2.31(m, 5H), 2.30-2.15(m, 4H) ,2.04-1.86(m,5H),1.82(t,J=24.9Hz,1H),1.77-1.69(m,1H),1.68-1.57(m,2H),1.39(d,J=2.9Hz,6H ), 0.58(s,3H).HRMS(ESI)(m/z)C ₃₄ H ₄₃ N ₂ O ₂ [M+H] ⁺ : Calc. 511.7174, Found 511.7173.

β-tert-butylamine 43B and α-tert-butylamine 43A

β-tert-butylamine 43B: The crude mixture was purified by flash chromatography (silica gel, eluent: 50:1 EtOAc:triethylamine) to afford β-tert-butylamine 43B (3.4 mg, 60%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.49(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.3Hz,1H),7.62 (d,J=5.9Hz,1H),7.59(d,J=8.3Hz,1H),5.75(d,J=2.0Hz,1H),5.29(m,1H),3.14(dd,J=10.8, 10.8Hz, 1H), 2.52(dd, J＝11.7, 8.8Hz, 1H), 2.32–2.38(m, 2H), 2.13-2.26(m, 5H), 2.01-2.08(m, 2H), 1.94(dd ,J＝17.6,5.4Hz,1H),1.84-1.87(m,3H),1.62-1.74(m,3H),1.25(br s,9H).HRMS(ESI)(m/z)C ₃₂ H ₄₁ N ₂ O[M+H] ⁺ : calculated value 469.3219, measured value 469.3265.

β-Aziridine 78B and α-Aziridine 78A

To a solution of ketone 13 (6 mg, 0.0146 mmol) in methanol (0.5 mL) was added 2-chloroethylamine hydrochloride (5.1 mg, 0.0437 mmol) followed by triethylamine (0.006 mL, 0.0437 mmol). The mixture was stirred at room temperature for 15 minutes. Glacial acetic acid (0.0025 mL, 0.0437 mmol) was added, and the mixture was stirred at room temperature for 20 minutes. The mixture was cooled to 0°C and sodium cyanoborohydride (3.2 mg, 0.0510 mml) was added. The reaction was allowed to warm to room temperature over 16 hours, then quenched with saturated ammonium chloride solution (5 mL). The mixture was extracted with ethyl acetate (3 x 8 mL). The combined organic fractions were dried over anhydrous magnesium sulfate, filtered and concentrated. The crude product was purified by silica gel chromatography (ethyl acetate, 2% triethylamine as eluent) to afford the desired β-aziridine 78B (4.5 mg, 72% yield).

¹ H NMR (500MHz, CDCl ₃ )δ=9.22(s,1H),8.48(d,J=5.87Hz,1H),7.79(s,1H),7.75(d,J=8.80Hz,1H),7.63 (d,J=5.38Hz,1H),7.59(d,J=8.80Hz,1H),5.74(d,J=1.96Hz,1H),5.25(m,1H),3.34(m,2H),3.14 (dd,J=10.27,10.27Hz,1H),2.75(m,3H),2.51(dd,J=11.25,8.31,1H),2.44(m,1H),2.29-2.37(m,3H),2.12 -2.27(m,3H),1.81-2.08(m,3H),1.54-1.74(m,4H),1.15(m,1H),1.05(m,1H),0.55(s,3H).HRMS(ESI )(m/z)C ₃₀ H ₃₅ N ₂ O[M+H] ⁺ : calculated value 439.2749 found value 439.2721.

β-Hydroxyproline 65B and α-Hydroxyproline 65A

Under Method B conditions, ketone 13 is reacted with hydroxyproline methyl ester. The crude mixture was dissolved in THF:MeOH:1M LiOH in _H2O =3:3:1 and stirred at 55°C for 1.5 hours. The crude mixture was roughly concentrated and sodium acetate buffer pH 3.7 was applied, then extracted three times with chloroform. The crude mixture was purified by preparative TLC (eluent: 5:1 _CHCl3 :MeOH) to afford β-hydroxyproline 65B (3.7 mg, 58% for two steps).

¹ H NMR (500MHz, CDCl ₃ ) shift = 9.22 (br.s., 1H), 8.47 (br.s., 1H), 7.77 (br.s., 1H), 7.75 (d, J = 8.2Hz, 1H), 7.63(br.s., 1H), 7.57(d, J=8.2Hz, 1H), 5.77(s, 1H), 5.30(br.s., 1H), 4.47(br.s., 1H ),4.21-4.08(m,1H),4.01-3.90(m,0H),3.55(br.s.,1H),3.19-3.11(m,1H),3.12(t,J＝8.8Hz,1H) ,2.51-2.44(m,J=10.6,10.6Hz,1H),2.44-2.37(m,2H),2.35(d,J=17.6Hz,2H),2.31-2.14(m,6H),2.11(dd ,J=6.2,13.2Hz,1H),2.06-1.96(m,2H),1.92(dd,J=4.7,17.6Hz,1H),1.87-1.68(m,3H),0.53(s,3H). HRMS(ESI)(m/z) C ₃₃ H ₃₉ N ₂ O ₄ [M+H] ⁺ : Calculated 527.2904, Found 527.2921.

α-Dimethylamine 14A and β-Dimethylamine 14B

The crude mixture was sequentially purified by flash chromatography (silica gel, eluent: 20:1 EtOAc:2M _NH3 in MeOH) to afford α-dimethylamine 14A (2.2 mg, 68%). ¹ H NMR (600MHz, C ₆ D ₆ ) Shift=9.26(s,1H),8.56(d,J=5.9Hz,1H),7.44-7.39(m,1H),7.36(d,J=8.2Hz, 1H),7.21-7.20(m,1H),7.20(d,J=5.9Hz,1H),5.68-5.65(m,1H),5.15-5.11(m,1H),2.72-2.66(m,J= 10.0Hz, 1H), 2.59(dd, J＝8.8, 11.2Hz, 1H), 2.34(tt, J＝2.9, 12.1Hz, 1H), 2.16(td, J＝3.2, 16.0Hz, 1H), 2.09( s,6H),2.13-1.92(m,8H),1.85(ddd,J＝5.0,9.0,13.6Hz,1H),1.73(dt,J＝5.3,12.3Hz,1H),1.72-1.66(m, 2H),1.60-1.57(m,1H),1.57-1.49(m,1H),1.20(dq,J=4.1,12.3Hz,1H),0.40(s,3H).HRMS(ESI)(m/z )C ₃₀ H ₃₇ N ₂ O[M+H] ⁺ : calculated value 441.2900, measured value 441.2909.

α-methylamine 24A and β-methylamine 24B

The crude mixture was purified by preparative TLC (silica gel, eluent: 10:1 EtOAc:2M _NH3 in MeOH) to afford α-monomethylamine 24A (ca. 1.2 mg, 37%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.81(s,1H),7.77(d,J=8.8Hz,1H),7.64 (d, J=5.4Hz, 1H), 7.61(dd, J=1.0, 8.8Hz, 1H), 5.76(s, 1H), 5.29(d, J=2.4Hz, 1H), 3.16(t, J= 10.0Hz,1H),2.61-2.50(m,3H),2.49(s,3H),2.43-2.30(m,3H),2.30-2.15(m,4H),2.13-1.99(m,2H),1.95 (dd,J=5.4,17.6Hz,1H),1.88(dq,J=4.9,11.7Hz,1H),1.79-1.60(m,3H),1.19(dq,J=4.4,12.7Hz,1H), 0.56(s,3H).HRMS(ESI)(m/z)C ₂₉ H ₃₅ N ₂ O[M+H] ⁺ : Calculated 427.2744, Found 427.2759.

α-primary amine 62A and β-primary amine 62B

The crude mixture was purified by preparative TLC (silica gel, eluent: 25:1 EtOAc:2M _NH3 in MeOH) to afford α-primary amine 62A (3.7 mg, 38%) and β-primary amine 62B (2.5 mg , 26%).

α-Primary amine 62A: ¹ H NMR (500MHz, CDCl ₃ ) shift=9.22(s, 1H), 8.48(d, J=5.9Hz, 1H), 7.79(s, 1H), 7.75(d, J=8.8 Hz,1H),7.62(d,J=5.3Hz,1H),7.59(dd,J=1.2,8.2Hz,1H),5.74(s,1H),5.27(d,J=2.9Hz,1H), 3.14(t, J=10.0Hz, 1H), 2.85(tt, J=3.2, 11.7Hz, 1H), 2.51(dd, J=8.5, 11.4Hz, 1H), 2.40-2.28(m, 3H), 2.27 -2.12(m,4H),2.10-1.96(m,3H),1.93(dd,J＝5.3,17.6Hz,1H),1.92-1.81(m,2H),1.76-1.62(m,2H),1.22 (dtd,J＝4.1,11.7,13.5Hz,1H),0.53(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₃ N ₂ O[M+H] ⁺ : calculated value 413.2587, measured Value 413.2590.

β-Primary amine 62B: ¹ H NMR (500MHz, CDCl ₃ ) shift=9.22(s, 1H), 8.48(d, J=5.9Hz, 1H), 7.79(s, 1H), 7.75(d, J=8.2 Hz,1H),7.62(d,J=5.3Hz,1H),7.59(dd,J=1.5,8.5Hz,1H),5.73(d,J=1.2Hz,1H),5.25(d,J=2.9 Hz,1H),3.47(td,J=3.7,7.9Hz,1H),3.13(t,J=10.0Hz,1H),2.58(dt,J=5.3,14.1Hz,1H),2.51(dd,J =8.5,11.4Hz,1H),2.39-2.21(m,5H),2.17(dq,J=5.3,9.4Hz,1H),2.13(ddd,J=2.3,4.7,15.8Hz,1H),2.10- 1.99(m,2H),1.93(dd,J=5.3,17.0Hz,1H),1.86(dq,J=5.3,12.3Hz,1H),1.82(ddd,J=1.8,4.1,13.5Hz,1H) ,1.78-1.66(m,3H),0.54(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₃ N ₂ O[M+H] ⁺ : calculated value 413.2587, found value 413.2599.

Morpholine 15A and Morpholine 15B

The crude mixture was purified by preparative TLC (silica gel, eluent: 40:1 EtOAc:MeOH) to afford α-morpholine 15A (ca. 1.5 mg, 38%). ¹ H NMR (600MHz, CDCl ₃ ) Shift=9.23(s,1H),8.49(d,J=5.4Hz,1H),7.80(s,1H),7.76(d,J=8.3Hz,1H),7.63 (d,J=5.4Hz,1H),7.60(d,J=8.3Hz,1H),5.74(s,1H),5.29(d,J=2.9Hz,1H),3.73(br.s.,4H ), 3.15(t, J=10.0Hz, 1H), 2.60(br.s., 4H), 2.52(dd, J=8.5, 11.5Hz, 2H), 2.46-2.29(m, 3H), 2.29-2.13 (m,4H),2.12-1.99(m,2H),1.94(dd,J=5.1,17.3Hz,1H),1.94-1.81(m,3H),1.73(td,J=8.2,12.3Hz,1H ), 1.70-1.63 (m, 1H), 1.38 (dq, J=4.4, 12.2Hz, 1H), 0.54 (s, 3H). HRMS (ESI) (m/z) C ₃₂ H ₃₉ N ₂ O ₂ [ M+H] ⁺ : calculated value 483.3006, measured value 483.3000.

α-Pyrrolidine 19A and β-Pyrrolidine 19B

The crude mixture was purified by preparative TLC (silica gel, eluent: 20:10:3 EtOAc:hexanes:2M _NH3 in MeOH) to afford α-pyrrolidine 19A (2.5 mg, 55%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.2Hz,1H),7.62 (d, J=5.3Hz, 1H), 7.59(d, J=8.8Hz, 1H), 5.72(s, 1H), 5.27(d, J=2.9Hz, 1H), 3.14(t, J=10.0Hz ,1H),2.63(br.s.,4H),2.52(dd,J＝8.8,11.2Hz,1H),2.42-2.29(m,3H),2.28-2.15(m,5H),2.12(d, J＝12.3Hz, 1H), 2.10-2.00(m, 2H), 1.93(dd, J＝5.3, 17.0Hz, 1H), 1.90-1.83(m, 2H), 1.80(br.s., 4H), 1.72(td,J=8.8,12.9Hz,1H),1.63(br.s.,1H),1.37(dq,J=3.5,11.7Hz,1H),0.53(s,3H).HRMS(ESI)( m/z) C ₃₂ H ₃₉ N ₂ O[M+H] ⁺ : calculated value 467.3057, found value 467.3064.

α-azetidine 18A and β-azetidine 18B

The crude mixture was purified by preparative TLC (silica gel, eluent: 1:1 EtOAc:MeOH) to afford α-azetidine 18A (ca. 1.5 mg, 38%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.4Hz,1H),7.80(s,1H),7.77(d,J=8.8Hz,1H),7.64 (d,J=5.9Hz,1H),7.60(d,J=8.8Hz,1H),5.74(s,1H),5.28(br.s.,1H),3.24(br.s.,4H), 3.16(t, J=9.8Hz, 1H), 2.54(dd, J=8.8, 11.2Hz, 1H), 2.42-2.30(m, 3H), 2.30-2.13(m, 5H), 2.12-2.00(m, 2H), 1.95(dd, J=5.4, 18.1Hz, 1H), 1.93-1.78(m, 3H), 1.74(td, J=8.3, 12.2Hz, 1H), 1.67-1.54(m, 3H), 1.11 (q,J=12.2Hz,1H),0.55(s,3H).HRMS(ESI)(m/z)C ₃₁ H ₃₇ N ₂ O[M+H] ⁺ : calculated value 453.2906, measured value 453.2900.

α-tert-butylamine 43A and β-tert-butylamine 43B

The crude mixture was purified by flash chromatography (silica gel, eluent: 10:1 _CHCl3 :i-PrOH) to afford α-tert-butylamine 43A (2.4 mg, 42%) and β-tert-butylamine 43B (1.6 mg, 28%) .

α-tert-butylamine 43A: ¹ H NMR (500MHz, CDCl ₃ ) shift=9.22(br.s.,1H),8.48(d,J=5.9Hz,1H),7.78(s,1H),7.75(d,J =8.8Hz,1H),7.62(d,J=5.3Hz,1H),7.58(dd,J=1.2,8.2Hz,1H),5.73(s,1H),5.28(d,J=2.3Hz,1H ), 3.14(t, J=9.7Hz, 1H), 2.49(dd, J=8.8, 11.2Hz, 1H), 2.46-2.40(m, 1H), 2.39-2.29(m, 3H), 2.28-2.12( m,5H),2.08-1.99(m,1H),1.93(dd,J=5.3,17.0Hz,1H),1.83(dq,J=5.0,12.2Hz,2H),1.76-1.60(m,3H) ,1.55(br.s.,9H),1.26-1.18(m,1H),0.54(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₄₁ N ₂ O[M+H] ⁺ : The calculated value is 469.3213, and the measured value is 469.3223.

β-tert-butylamine 43B: ¹ H NMR (500MHz, CDCl ₃ ) shift=9.22(s, 1H), 8.48(d, J=5.3Hz, 1H), 7.78(s, 1H), 7.75(d, J=8.8Hz ,1H),7.62(d,J=5.9Hz,1H),7.59(d,J=9.4Hz,1H),5.73(br.s.,1H),5.25(br.s.,1H),3.38- 3.23(m,1H),3.13(t,J=10.0Hz,1H),2.57-2.48(m,1H),2.49(dd,J=8.5,10.9Hz,1H),2.40-2.27(m,2H) ,2.25-2.08(m,4H),2.07-1.98(m,2H),1.93(dd,J=5.3,17.6Hz,2H),1.84(dq,J=5.3,12.3Hz,1H),1.76-1.66 (m,2H),1.65-1.47(m,3H),1.42-0.94(br.s.,9H),0.54(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₄₁ N ₂ O [M+H] ⁺ : calculated value 469.3213, measured value 469.3225.

α-Hydroxyazetidine 70A and β-Hydroxyazetidine 70B

The crude mixture was purified by preparative TLC (silica gel, eluent: 2:3 EtOAc:MeOH) to afford α-hydroxyazetidine 70A (1.2 mg, 30%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.4Hz,1H),7.80(s,1H),7.77(d,J=8.3Hz,1H),7.64 (d, J=5.9Hz, 1H), 7.60(d, J=8.3Hz, 1H), 5.77(s, 1H), 5.32(d, J=2.4Hz, 1H), 4.66-4.55(m, 1H) ,3.94(br.s.,2H),3.34(br.s.,2H),3.16(t,J=9.8Hz,1H),2.53(dd,J=8.5,11.5Hz,1H),2.41(dd ,J=15.6,28.3Hz,2H),2.34(dt,J=4.9,11.2Hz,1H),2.28(t,J=11.2Hz,1H),2.25-2.14(m,3H),2.10-1.98( m,2H),1.95(dd,J=5.4,17.6Hz,1H),1.96-1.89(m,1H),1.86(dd,J=5.4,12.2Hz,1H),1.84-1.76(m,2H) ,1.74(td,J=8.4,12.4Hz,1H),1.65(dt,J=7.8,10.5Hz,1H),1.40-1.27(m,1H),0.55(s,3H).HRMS(ESI)( m/z) C ₃₁ H ₃₇ N ₂ O ₂ [M+H] ⁺ : calculated value 469.2850, found value 469.2872.

α-Hydroxymethylazetidine 69A and β-Hydroxymethylazetidine 69B

The crude mixture was purified by preparative TLC (silica gel, eluent: 1:1 EtOAc:MeOH) to afford α-hydroxymethylazetidine 69A (2.3 mg, 53%). ¹ H NMR (600MHz, CDCl ₃ ) shift=9.23(s,1H),8.49(d,J=5.9Hz,1H),7.80(s,1H),7.76(d,J=8.2Hz,1H),7.63 (d,J=5.9Hz,1H),7.59(d,J=8.8Hz,1H),5.76(s,1H),5.30(d,J=2.9Hz,1H),3.65-3.35(m,4H) ,3.15(t,J=10.0Hz,1H),2.52(dd,J=8.5,11.4Hz,1H),2.40(dd,J=16.4,25.8Hz,2H),2.33(dt,J=4.1,11.7 Hz, 1H), 2.26(t, J=11.4Hz, 1H), 2.24(m, 3H), 2.09-2.00(m, 1H), 1.98(br.s., 1H), 1.94(dd, J=5.0 ,17.3Hz,1H),1.92-1.77(m,4H),1.73(td,J＝8.2,12.9Hz,1H),1.67-1.59(m,1H),1.58-1.50(br.s.,3H) ,1.39-1.28(m,1H),0.58-0.51(s,3H).HRMS(ESI)(m/z)C ₃₂ H ₃₉ N ₂ O ₂ [M+H] ⁺ : calculated value 483.3006, measured value 483.3000 .

α-aminoethylsulfonamide 71A and β-aminoethylsulfonamide 71B

The crude mixture was purified by preparative TLC (silica gel, eluent: 100% EtOAc) to give β-aminoethylsulfonamide 71B (0.7 mg, 15%) and α-aminoethylsulfonamide 71A (1.1 mg, 24% ).

β-Aminoethylsulfonamide 71B: ¹ H NMR (500MHz, CD ₃ OD) shift = 9.21-9.17 (m, 1H), 8.37 (d, J = 5.9Hz, 1H), 7.97 (s, 1H), 7.88 (d,J=8.3Hz,1H),7.79(d,J=5.4Hz,1H),7.74(dd,J=1.5,8.8Hz,1H),5.74-5.69(m,1H),5.28-5.22( m,1H),3.26-3.18(m,J＝9.8Hz,1H),3.14-3.00(m,4H),2.59-2.38(m,4H),2.37-2.26(m,2H),2.23-2.06( m,3H),2.03-1.86(m,5H),1.85-1.76(m,1H),1.76-1.59(m,3H),0.57(s,3H) _.HRMS (ESI)(m/z)C H ₃₈ N ₃ O ₃ S[M+H] ⁺ : calculated value 520.2628, measured value 520.2640.

α-Aminoethylsulfonamide 71A: ¹ H NMR (500MHz, CD ₃ OD) shift = 9.19(s, 1H), 8.37(d, J = 5.9Hz, 1H), 7.97(s, 1H), 7.88(d ,J=8.8Hz,1H),7.79(d,J=5.9Hz,1H),7.74(dd,J=1.7,8.6Hz,1H),5.77-5.71(m,1H),5.30-5.24(m, 1H), 3.29-3.24(m, 2H), 3.23(dd, J=9.0, 11.0Hz, 1H), 3.13(dt, J=2.7, 6.7Hz, 2H), 2.73(tt, J=3.1, 12.0Hz ,1H),2.50(dd,J=8.6,11.5Hz,1H),2.47-2.38(m,2H),2.39-2.34(m,1H),2.32(d,J=11.2Hz,1H),2.30- 2.22(m,2H),2.17(dtd,J＝5.9,9.3,14.7Hz,2H),2.10-2.03(m,1H),2.01(s,1H),2.00-1.92(m,1H),1.90( dd,J=6.4,17.1Hz,1H),1.72(td,J=8.3,12.3Hz,1H),1.68-1.60(m,2H),1.18(dq,J=4.4,12.2Hz,1H),0.56 (s,3H).HRMS(ESI)(m/z)C ₃₀ H ₃₈ N ₃ O ₃ S[M+H] ⁺ : calculated value 520.2628, found value 520.2643.

α-Hydroxyaminomethyloxetane 72A and β-Hydroxyaminomethyloxetane 72B

The crude mixture was purified by preparative TLC (silica gel, eluent: 100% EtOAc) to afford α-hydroxyaminomethyloxetane 72A (1.5 mg, 34%). ¹ H NMR (500MHz, CD ₃ OD) shift=9.19(s,1H),8.38(d,J=5.4Hz,1H),7.97(s,1H),7.88(d,J=8.3Hz,1H), 7.79(d,J=5.9Hz,1H),7.74(dd,J=1.5,8.8Hz,1H),5.84-5.78(m,1H),5.35-5.30(m,1H),4.64(d,J= 7.3Hz, 2H), 4.57(dd, J＝3.7, 7.1Hz, 2H), 3.48-3.40(m, 2H), 3.24(dd, J＝9.0, 11.0Hz, 2H), 2.54-2.48(m, J ＝8.8Hz,1H),2.48-2.40(m,3H),2.40-2.24(m,4H),2.24-2.13(m,3H),2.01-1.85(m,4H),1.80-1.64(m,2H ), 1.46 (dq, J=4.9, 12.2Hz, 1H), 0.57 (s, 3H).HRMS (ESI) (m/z) C ₃₂ H ₃₉ N ₂ O ₃ [M+H] ⁺ : calculated value 499.2955 , the measured value is 499.2933.

β-PEG Amine 75B and α-PEG Amine 75A

β-PEG amine 75B: The crude mixture was purified by preparative TLC (silica gel, eluent: 5:5: ₁ EtOAc:dichloromethane:2M NH in MeOH) to afford α-PEG amine 75B (1.0 mg, 18%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.81(s,1H),7.77(d,J=8.3Hz,1H),7.64 (d,J=5.9Hz,1H),7.61(dd,J=1.7,8.5Hz,1H),5.72(d,J=1.5Hz,2H),5.25(dd,J=2.2,5.1Hz,1H) ,3.71-3.64(m,6H),3.62(t,J=5.4Hz,2H),3.58(dd,J=3.7,5.6Hz,2H),3.41(s,3H),3.19-3.12(m,J =10.7Hz, 1H), 3.11(t, J=3.9Hz, 1H), 2.79(t, J=5.4Hz, 2H), 2.56(t, J=16.1Hz, 1H), 2.52(dd, J=8.3 ,11.2Hz,1H),2.42-2.29(m,3H),2.27-2.13(m,2H),2.12-2.01(m,2H),2.02(dd,J=3.7,13.9Hz,1H),1.95( br.s., 2H), 1.86(dq, J=5.4, 12.2Hz, 1H), 1.80-1.73(m, 1H), 1.71(dd, J=3.2, 11.5Hz, 1H), 1.68-1.58(m ,2H),0.56(s,3H).HRMS(ESI)(m/z)C ₃₅ H ₄₇ N ₂ O ₄ [M+H] ⁺ : Calculated 559.3530, Found 559.3545.

α-PEGamine 75A: The crude mixture was purified by preparative TLC (silica gel, eluent: 100:5:1 EtOAc:MeOH:triethylamine) to afford α-PEGamine 75A (1.1 mg, 20%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.81(s,1H),7.77(d,J=8.8Hz,1H),7.64 (d,J=5.9Hz,1H),7.61(dd,J=1.5,8.8Hz,1H),5.75(d,J=2.0Hz,1H),5.28(dd,J=2.2,5.1Hz,1H) ,3.70-3.65(m,7H),3.64(t,J=5.4Hz,2H),3.61-3.55(m,2H),3.41(s,3H),3.16(dd,J=9.0,10.5Hz,1H ), 2.87(t, J=5.1Hz, 2H), 2.67(t, J=11.5Hz, 1H), 2.54(dd, J=8.3, 11.7Hz, 1H), 2.38(d, J=16.1Hz, 3H ),2.24(d,J=12.2Hz,2H),2.22-2.14(m,2H),2.11-2.02(m,2H),1.95(dd,J=5.4,16.6Hz,1H),1.88(dq, J＝5.4,12.2Hz,1H),1.78-1.69(m,2H),1.68-1.62(m,1H),1.27-1.19(m,1H),0.55(s,3H).HRMS(ESI)(m /z) C ₃₅ H ₄₇ N ₂ O ₄ [M+H] ⁺ : calculated value 559.3530, found value 559.3542.

α-Methylsulfonamide 73A

The crude mixture was purified by preparative TLC (silica gel, eluent: 40:1 MeOH:dichloromethane) to afford α-methylsulfonamide 73A (2.0 mg, 82%). ¹ H NMR (500MHz, CDCl ₃ ) Shift = 9.24(br.s., 1H), 8.51(d, J=4.9Hz, 1H), 7.80(s, 1H), 7.77(d, J=8.3Hz, 1H ),7.64(d,J=5.9Hz,1H),7.60(dd,J=1.5,8.3Hz,1H),5.78(d,J=1.5Hz,1H),5.36-5.29(m,1H),4.24 (d, J=7.8Hz, 1H), 3.53(tdt, J=3.9, 7.7, 11.7Hz, 1H), 3.20-3.11(m, J=10.7Hz, 1H), 3.04(s, 3H), 2.51( dd,J=8.3,11.7Hz,1H),2.37(d,J=16.6Hz,3H),2.28(br.s.,2H),2.25-2.10(m,4H),2.10-2.00(m,1H ),1.96(dd,J=5.4,17.6Hz,1H),1.88(dt,J=5.4,11.7Hz,1H),1.85(t,J=12.2Hz,1H),1.80-1.66(m,2H) , 1.47-1.35 (m, J=4.4Hz, 1H), 0.55 (s, 3H); HRMS (ESI) (m/z) C ₂₉ H ₃₅ N ₂ O ₃ S[M+H] ⁺ : Calculated 491.2363 , the measured value is 491.2387.

β-Methylsulfonamide 73B

The crude mixture was purified by preparative TLC (silica gel, eluent: 40:1 MeOH:dichloromethane) to afford β-methylsulfonamide 73B (1.7 mg, 90%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.80(s,1H),7.78(d,J=8.8Hz,1H),7.64 (d,J=5.9Hz,1H),7.60(dd,J=1.5,8.3Hz,1H),5.79(d,J=1.5Hz,1H),5.35-5.30(m,1H),4.31(d, J＝6.3Hz, 1H), 4.00(quind, J＝3.6, 6.7Hz, 1H), 3.16(dd, J＝9.0, 10.5Hz, 1H), 3.03(s, 3H), 2.52(dd, J＝8.5 ,11.5Hz,1H),2.41(t,J=16.1Hz,1H),2.39(br.s.,2H),2.32-2.26(m,2H),2.26-2.14(m,3H),2.12-1.99 (m,2H),1.96(dd,J=5.1,17.3Hz,2H),1.86(dq,J=5.4,12.2Hz,1H),1.83-1.72(m,3H),0.56(s,3H); HRMS(ESI)(m/z) C ₂₉ H ₃₅ N ₂ O ₃ S[M+H] ⁺ : Calculated 491.2363, Found 491.2376.

α-Methyl-methylsulfonamide 76A

The crude mixture was purified by preparative TLC (silica gel, eluent: 40:1 MeOH:dichloromethane) to afford α-methyl-methylsulfonamide 76A (0.7 mg, 57%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.24(s,1H),8.51(d,J=5.9Hz,1H),7.81(s,1H),7.78(d,J=8.3Hz,1H),7.64 (d,J=5.9Hz,1H),7.60(dd,J=1.5,8.3Hz,1H),5.79(s,1H),5.33(dd,J=2.2,5.6Hz,1H),3.98(tt, J=3.4,12.4Hz,1H),3.16(dd,J=9.0,10.5Hz,1H),2.89(s,3H),2.80(s,3H),2.52(dd,J=8.5,11.5Hz,1H ),2.44(ddd,J=2.7,4.1,16.6Hz,1H),2.36(br.s.,3H),2.28(d,J=10.7Hz,2H),2.21(dq,J=4.9,9.1Hz ,1H),2.14(t,J=12.7Hz,1H),2.10-2.00(m,0H),1.97(dd,J=5.4,17.6Hz,1H),1.93(dd,J=2.9,12.2Hz, 1H), 1.88(dd, J=5.4, 12.2Hz, 1H), 1.87-1.81(m, 1H), 1.80-1.60(m, 3H), 0.56(s, 3H).HRMS(ESI)(m/z )C ₃₀ H ₃₇ N ₂ O ₃ S[M+H] ⁺ : calculated value 505.2519, measured value 505.2537.

Example S4. Another possible route from isoquinoline compounds 10-12

A new route to isoquinoline 12 was designed. See Scenario 2-1 below. Triflation/Suzuki cross-coupling reactions were achieved on analogous substrates for the indicated reagents shown in the figure. See eg Nicolaou et al., J. Am. Chem. Soc. 2009, 131, 10587-10597.

Scheme 4-1.

Synthesis of Triflate 20

To a solution of monoketone 10 (200 mg, 584 μmol, 1.00 equiv) in THF (4 mL) was added NaHMDS (1M, 701 μL, 701 μmol, 1.20 equiv) dropwise at -78°C. After stirring for 1.5 h, _PhNTf2 (313 mg, 876 μmol, 1.50 equiv) in THF (2.5 mL) was cannulated and the reaction mixture was warmed to 0 °C. After another 30 minutes, saturated _NH4Cl solution (8 mL) was added to the stirred reaction mixture and diluted with EtOAc (10 mL). The layers were separated, the aqueous layer was extracted with EtOAc (2 x 6 mL), the combined organic layers were washed with brine (15 mL), dried _{over Na2SO4} , and concentrated under reduced pressure. The resulting residue was then purified by flash column chromatography (silica gel, eluent: 8:1→5:1 Hex:EtOAc) to afford triflate 20 (237 mg, 86%). ¹ H NMR (500MHz, CDCl ₃ ) shift=5.76(s,1H),5.67(br.s.,1H),5.32(dd,J=2.0,4.9Hz,1H),4.02-3.94(m,4H) ,2.67(dd,J=6.8,10.7Hz,1H),2.49(t,J=14.6Hz,1H),2.45(ddd,J=3.7,6.5,15.2Hz,1H),2.38-2.28(m,4H ), 2.17(ddd, J=1.5, 10.7, 12.7Hz, 1H), 2.12(d, J=13.2Hz, 1H), 2.10(dd, J=5.9, 17.6Hz, 1H), 1.98(dd, J= 2.7,13.4Hz,1H),1.88(ddd,J＝7.6,8.9,12.8Hz,1H),1.80(tdd,J＝2.4,4.8,12.7Hz,1H),1.74-1.63(m,2H),1.03 (s,3H).HRMS(ESI)(m/z)C ₂₂ H ₂₆ O ₆ F ₃ S[M+H] ⁺ : calculated value 475.1397, found value 475.1411.

Synthesis of 17,18-unsaturated isoquinolines 21 by Suzuki cross-coupling of triflate 20

To a solution of triflate 20 (1.00 equiv) and isoquinoline-7-boronic acid (3.00 equiv) in 1,4-dioxane and _H2O (10:1, 0.02M) was added _{K2CO 3} (3.00 equiv), and the solution was bubbled through inert Ar for 5 min. Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (0.05 equiv) was added and the reaction mixture was stirred at 80° C. for 1 hour. The mixture was cooled to room temperature, and saturated NaHCO ₃ solution was applied. The mixture was diluted with EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried _{over Na2SO4} , and concentrated under reduced pressure.

The crude mixture was purified by flash column chromatography (silica gel, eluent: 2:1 → 1:1 → 1:2 in hexane:EtOAc) to give 17,18-unsaturated isoquinoline 21 (490 mg, 84%) . ¹ H NMR (500MHz, CDCl ₃ ) shift = 9.23 (s, 1H), 8.49 (d, J = 5.4Hz, 1H), 7.94 (s, 1H), 7.85-7.81 (m, 1H), 7.80-7.75 ( m,1H),7.63(d,J=5.4Hz,1H),6.26(br.s.,1H),5.82(s,1H),5.40(d,J=3.4Hz,1H),4.08-3.90( m, 4H), 2.76(dd, J=7.1, 11.0Hz, 1H), 2.58(dt, J=5.4, 17.6Hz, 1H), 2.56-2.40(m, 3H), 2.40-2.28(m, 4H) ,2.16(d,J=13.2Hz,1H),2.02(dd,J=2.0,13.2Hz,1H),1.94(td,J=8.8,13.2Hz,1H),1.81(td,J=2.0,12.7 Hz,1H),1.76-1.67(m,2H),1.18(s,3H).HRMS(ESI)(m/z)C ₃₀ H ₃₂ NO ₃ [M+H] ⁺ : calculated value 454.2377, measured value 454.2366 .

Synthesis of Isoquinoline 12 from 17,18-Unsaturated Isoquinoline 21

To a solution of 17,18-unsaturated isoquinoline 21 (400 mg, 877 μmol, 1.0 equiv) in THF (36 mL) was added 10 wt% Pd/C (280 mg, 263 μmol, 0.30 equiv) and a _H2 balloon was installed. After 3 h, the reaction mixture was filtered through a pad of Celite and washed with 0.2M _NH3 in MeOH (40 mL), concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, eluent: 1:1→1:2 hexane:EtOAc) to provide isoquinoline 12 (325 mg, 80%). Spectral data are consistent with isoquinoline 12 constructed from 1-chloroisoquinoline adduct 11.

Example S5. Synthesis of Isoquinoline Analogs

Scheme 5-1.

Scheme 5-2.

Scheme 5-3.

Scheme 5-4.

Synthesis of 17,18-unsaturated 5-indazole 56 by Suzuki cross-coupling of triflate 20

_{K 2} CO ₃ (3.00 equiv) and inert Ar was bubbled through the solution for 5 min. Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (0.05 equiv) was added and the reaction mixture was stirred at 80° C. for 1 hour. The mixture was cooled to room temperature, and saturated NaHCO ₃ solution was applied. The mixture was diluted with EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried _{over Na2SO4} , and concentrated under reduced pressure.

The crude mixture was purified by flash column chromatography (silica gel, eluent: 1:3→1:1 EtOAc:hexanes) to afford 17,18-unsaturated 6-indazole 56 (13 mg, 70%). ¹ H NMR (500MHz, CDCl ₃ ) shift=8.04(s,1H),7.67(d,J=10.0Hz,1H),7.49(s,1H),7.27(d,J=10.0Hz,1H),6.12 (br.s.,1H),5.78(br.s.,1H),5.36(t,J=5.0Hz,1H),3.99(m,4H),2.72(m,1H),2.53-2.44(m ,3H),2.40(br.d.,J=10.0Hz,1H),2.36-2.25(m,4H),2.14(d,J=10.0Hz,1H),2.00(dd,J=10.0,5.0Hz ,1H),1.93-1.87(m,1H),1.79(m,1H),1.72-1.66(m,2H),1.10(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₁ N ₂ O ₃ [M+H] ⁺ : calculated value 443.5573, measured value 443.5571.

Synthesis of 17,18-unsaturated 6-indazole 59 by Suzuki cross-coupling of triflate 20

_{K 2} CO ₃ (3.00 equiv), and inert Ar was bubbled through the solution for 5 min. Pd(dppf)Cl ₂ ·CH ₂ Cl ₂ (0.05 equiv) was added and the reaction mixture was stirred at 80° C. for 1 hour. The mixture was cooled to room temperature, and saturated NaHCO ₃ solution was applied. The mixture was diluted with EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with brine, dried _{over Na2SO4} , and concentrated under reduced pressure.

The crude mixture was purified by flash column chromatography (silica gel, eluent: 1:4→3:1 EtOAc:hexanes) to afford 17,18-unsaturated 5-indazole 59 (5.9 mg, 65%). ¹ H NMR (500MHz, CDCl ₃ ) Shift = 10.04(br s, 1H), 8.05(s, 1H), 7.75(s, 1H), 7.46(ABq, J _AB = 8.8Hz, Δν = 33.5Hz, 2H) ,6.02(s,1H),5.79(s,1H),5.38(dd,J=3.4,3.4Hz,1H),3.94-4.01(m,4H),2.73(dd,J=10.7,6.8Hz,1H ),2.44-2.53(m,3H),2.40(d,12.2Hz,1H),2.28-2.36(m,3H),2.15(d,J＝13.2Hz,1H),2.03(par obs d,J＝ 11.2Hz, 1H), 2.01(parobs dd, J＝13.2, 2.4Hz, 1H), 1.91(dt, J＝12.21, 9.3Hz, 1H), 1.77-1.82(m, 1H), 1.66-1.73(m, 2H), 1.10(s,3H).HRMS(ESI)(m/z) C ₂₈ H ₃₁ N ₂ O ₃ [M+H] ⁺ : Calculated 443.2335, Found 443.4956.

β-dimethylaminomethylpyridine 46B and α-dimethylaminomethylpyridine 46A

β-Dimethylaminomethylpyridine 46B: Purification of the crude mixture by flash chromatography (silica gel, eluent: 10:1 EtOAc:2M _NH3 in MeOH) gave β-dimethylaminomethylpyridine 46B (5 mg, 55%). ¹ H NMR (500MHz, CDCl ₃ ) Shift = 8.58(s, 1H), 8.51(dd, J=1.5, 4.9Hz, 1H), 7.71(td, J=2.0, 7.8Hz, 1H), 7.26(dd, J＝4.9, 7.8Hz, 1H), 5.71(s, 1H), 5.24(dd, J＝2.4, 4.4Hz, 1H), 3.88-3.80(m, 2H), 2.81(t, J＝9.0Hz, 1H ),2.45(dt,J＝6.3,13.7Hz,1H),2.46-2.37(m,1H),2.30(br.s.,6H),2.26-2.19(m,3H),2.17-2.07(m, 5H), 1.92(dd, J=2.9, 13.7Hz, 2H), 1.79(dddd, J=4.9, 8.3, 10.3, 13.2Hz, 1H), 1.74-1.59(m, 4H), 1.39(ddt, J= 4.4,9.8,12.7Hz,1H),0.80(s,3H).HRMS(ESI)(m/z)C ₂₇ H ₃₈ N ₃ O[M+H] ⁺ : calculated value 420.3009, found value 420.2999.

β-Dimethylamine 17,18-unsaturated amide pyridine 49B and α-dimethylamine 17,18-unsaturated amide pyridine 49A

β-Dimethylamine 17,18-Unsaturated amidopyridine 49B: Purification of the crude mixture by flash chromatography (silica gel, eluent: 8:1 EtOAc:2M _NH3 in MeOH) gave β-dimethylamine 17 , 18-Unsaturated amidopyridine 49B (2.1 mg, 44%). ¹ H NMR (500MHz, CDCl ₃ ) shift = 8.58 (d, J = 2.4Hz, 1H), 8.36 (dd, J = 1.0, 4.9Hz, 1H), 8.23 (td, J = 2.0, 8.3Hz, 1H) ,7.49(s,1H),7.29(dd,J=4.9,8.8Hz,1H),6.57(br.s.,1H),5.73(s,1H),5.33(dd,J=2.0,5.4Hz, 1H),2.66-2.56(m,2H),2.54(dd,J＝3.2,6.6Hz,1H),2.51-2.32(m,5H),2.28(br.s.,6H),2.20(ddd,J =1.2,11.0,12.7Hz,1H),2.10(td,J=6.4,13.1Hz,2H),1.97-1.89(m,3H),1.76(dt,J=7.8,11.2Hz,1H),1.66- 1.55(m,1H),1.14(s,3H).HRMS(ESI)(m/z)C ₂₇ H ₃₄ N ₃ O ₂ [M+H] ⁺ : Calculated 432.2646, Found 432.2649.

β-Dimethylamine 17,18-Unsaturated Phthalazine 55B and α-Dimethylamine 17,18-Unsaturated Phthalazine 55A

β-Dimethylamine 17,18-unsaturated phthalazine 55B: Purification of the crude mixture by flash chromatography (silica gel, eluent: 9:1 EtOAc:2M _NH3 in MeOH) gave β-dimethylamine 17, 18-Unsaturated Phthalazine 55B (5.5 mg, 73%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.50(s,2H),7.97-8.03(m,1H),7.89(d,J=4.39Hz,2H),6.34-6.39(m,1H),5.77- 5.83(m,1H),5.32-5.43(m,1H),2.72(dd,J=11.23,6.84Hz,1H),2.38-2.50(m,4H),2.47(br.m.,6H),2.24 -2.30(m,3H),2.09-2.20(m,2H),2.03(d,J＝10.25Hz,2H),1.88-1.99(m,2H),1.75-1.86(m,2H),1.17(s ,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₃ N ₃ O[M+H] ⁺ : Calculated 440.5998, Found 440.5995.

β-dimethylamine 17,18-unsaturated 6-indazole 58B and α-dimethylamine 17,18-unsaturated 6-indazole 58A

β-Dimethylamine 17,18-unsaturated 6-indazole 58B: Purification of the crude mixture by flash chromatography (silica gel, eluent: 9:1 EtOAc:2M _NH3 in MeOH) afforded β-dimethylamine Amine 17,18-unsaturated 6-indazole 58B (3.9 mg, 73%). ¹ HNMR (500MHz, CDCl ₃ ) shift = 8.03(s, 1H), 7.67(d, J = 8.30Hz, 1H), 7.49(s, 1H), 7.25-7.28(m, 1H), 6.12(t, J =2.44Hz,1H),5.75(s,1H),5.33(t,J=3.42Hz,1H),3.22-3.38(m,1H),2.71(dd,J=11.23,6.84Hz,1H),2.44 -2.50(m,4H),2.41(br.m.,6H),2.22-2.31(m,3H),2.09-2.20(m,2H),2.04(d,J＝2.93Hz,2H),1.78( td,J＝11.35,7.08Hz,2H),1.52-1.64(m,1H),1.07-1.15(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₃ N ₃ O[M+H ] ⁺ : calculated value 428.5891, measured value 428.5889.

β-dimethylamine 17,18-unsaturated 5-indazole 61B and α-dimethylamine 17,18-unsaturated 5-indazole 61A

β-Dimethylamine 17,18-unsaturated 5-indazole 61B: Purification of the crude mixture by flash column chromatography (silica gel, eluent: 9:1 EtOAc:2M _NH3 in MeOH) afforded β-di Methylamine 17,18-unsaturated 5-indazole 61B (2.5 mg, 70%). ¹ H NMR (500MHz, CDCl ₃ ) Shift = 8.05(s, 1H), 7.75(s, 1H), 7.47(ABq, J _AB = 8.8Hz, Δν = 33.5Hz, 2H), 6.02(dd, J = 2.0 ,2.0Hz,1H),5.74(s,1H),5.33(dd,J=3.4,3.4Hz,1H),2.72(dd,J=11.2,6.8Hz,1H),2.43-2.48(m,5H) ,2.37-2.41(m,2H),2.26-2.35(m,8H),2.11(m,2H),2.04(d,J＝6.8Hz,1H),1.88–1.98(m,3H),1.77(m ,1H),1.10(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₄ N ₃ O[M+H] ⁺ : Calculated 428.2702, Found 428.2653.

Synthesis of β-dimethylaminopyridine 50B

To a solution of β-dimethylamine 17,18-unsaturated amidopyridine 49B (ca. 1.2 mg) in MeOH (300 μL) was added Mg (ca. 1 mg) and stirred at room temperature for 48 h. _H2O (700 μL) was added to the reaction mixture and diluted with EtOAc (700 μL). The aqueous phase was extracted with EtOAc (2 x 0.5 mL), the combined organic phases were washed with brine (0.5 mL), dried _{over Na2SO4} , and concentrated under reduced pressure. The residue was purified by HPLC (Eclipse XDB-C8 column, 9.4 mm x 25 cm; gradient = 0% → 35% MeCN (0.1% formic acid): H ₂ O (0.1% formic acid) over 30 minutes) to afford β-dimethylamine Ampyridine 50B (ca. 0.3 mg, 25%). Due to the low volume, only diagnostic peaks were assigned. ¹ H NMR (500MHz, CDCl ₃ ) shift = 8.56 (br.s, 1H), 8.37 (d, J = 3.4Hz, 1H), 8.21 (d, J = 8.3Hz, 1H), 7.09 (br.s. ,1H),5.81(s,1H),5.39-5.33(m,1H),2.78(br.s.,6H),0.83(s,3H).HRMS(ESI)(m/z)C ₂₇ H ₃₅ N ₃ O ₂ [M+H] ⁺ : calculated value 434.2802, measured value 434.2815.

Synthesis of Phthalazine 6-Trifluoromethanesulfonate 51

N-Phenyl-bis(trifluoromethanesulfonimide) (1.73 g, 4.83 mmol, 1.2 eq), Et ₃ N (0.9 mL, 6.04 mmol, 1.5 eq) and DMAP (cat.) were added to 6- Phthalazinol (588.4 mg, 4.26 mmol, 1.0 equiv) in CHCl ₃ . The mixture was warmed to 60°C and stirred for 3 hours. The reaction was cooled to room temperature, quenched with saturated _NaHCO3 and _CH2Cl2 , and the layers _were separated. _The aqueous layer was extracted with _CH2Cl2 . The organic layers were combined, dried _{over Na2SO4} , and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (silica gel, eluent: 9:1 dichloromethane:MeOH) to afford phthalazine 6-triflate 51 (995 mg, 90%). ¹ H NMR (500MHz, CDCl ₃ ) shift = 9.68 (d, J = 3.91 Hz, 2H) 8.19 (d, J = 8.79 Hz, 1H) 7.96 (br.s., 1H) 7.84-7.89 (m, 1H) .HRMS(ESI)(m/z)C ₉ H ₆ F ₃ N ₂ O ₃ S[M+H] ⁺ : Calculated 279.2157, Found 279.2152.

Synthesis of Phthalazine 6-Trimethyltin 52

To a solution of phthalazine ₆ -triflate 52 (992 mg, 3.57 mmol, 1.0 eq) in _C6H6 was added LiCl (907 mg, 21.59 mmol, 6.0 eq), Pd( _PPh3 ) ₄ (412 mg, 0.3565 mmol, 0.1 equiv) and (Me3Sn _{)2 (} 0.78 mL, 3.743 mmol, 1.05 equiv). The solution was bubbled with argon in a sonicator for 10 minutes and the mixture was warmed to 105 °C and stirred for 1 hour. The reaction was cooled to room temperature, diluted with ethyl acetate and filtered through celite. The organic portion was washed with saturated NaHCO ₃ , dried over Na ₂ SO ₄ , and concentrated under reduced pressure. The crude mixture was purified by flash chromatography (silica gel, eluent: 1:1 EtOAc:hexanes) to afford the phthalazine 6-trimethyltin 52 (656 mg, 63%). ¹ H NMR (500MHz, CDCl ₃ ) shift = 9.54 (d, J = 4.39Hz, 2H) 8.12 (br.s., 1H) 8.08 (d, J = 7.81Hz, 1H) 7.92 (d, J = 7.81Hz ,1H)0.43(s,9H).HRMS(ESI)(m/z)C ₁₁ H ₁₅ N ₂ Sn[M+H] ⁺ : calculated value 293.9602, found value 293.9601.

Synthesis of 17,18-Unsaturated Phthalazine 53

To a solution of triflate 20 (20 mg, 42.15 μmol, 1.0 eq) in DMSO was added (trimethylstannyl)phthalazine 52 (31 mg, 105.40 μmol, 2.0 eq), CuCl (42 mg, 421.50 μmol , 10.0 equiv) and LiCl (18 mg, 421.50 mmol, 10.0 equiv). The mixture was deoxygenated four times by freeze-thaw method, and Pd( _PPh3 ) (5 mg, 4.22 μmol, 0.1 equiv) was added. The mixture was heated to 60°C and stirred for 1 hour. The reaction was quenched with 5% _NH4OH and ethyl acetate and the layers were separated. The aqueous layer was extracted with ethyl acetate. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure. The crude mixture was purified by flash chromatography (silica gel, eluent: 1:4→1:1→3:1 EtOAc:Hexane) to afford 17,18-unsaturated phthalazine 53 (16.3 mg, 85%). ¹ H NMR (500MHz, CDCl ₃ ) Shift = 9.51(d, J=5.0Hz, 2H) 8.02(d, J=5.0Hz, 1H), 7.91(s, 1H), 7.90(d, J=5.0Hz, 1H),6.37(br.s.,1H),5.80(br.s.,1H),5.37(br.m.,1H),3.98(m,4H),2.75(m,1H),2.59-2.52 (m,2H),2.50-2.42(m,3H),2.37-2.26(m,3H),2.14(d,J=15.0Hz,1H),2.00(dd,J=15.0,2.5Hz,1H), 1.93(m,1H),1.79(m,1H),1.72-1.66(m,2H),1.16(s,3H).HRMS(ESI)(m/z)C ₂₉ H ₃₁ N ₂ O ₃ [M+ H] ⁺ : calculated value 455.5680, measured value 455.5679.

Synthesis of 17,18-unsaturated amidopyridine 47

To a solution of triflate 20 (20 mg, 42.1 μmol, 1.0 equiv) and 3-aminopyridine (19.8 mg, 210 μmol, 5.0 equiv) in DMF (1 mL) was added triethylamine (12 μL, 84.3 mmol, 2.0 equiv) and Pd(dppf) _Cl2.CH2Cl2 (1.72 _mg , _2.10 mmol, 0.05 equiv). The reaction flask was filled with a CO balloon and the solution was purged for 5 min at room temperature. Then, the reaction mixture was heated to 85°C and stirred for 4 hours. The mixture was cooled to room temperature, EtOAc (3 mL) and _H2O were added. The layers were separated, the aqueous layer was extracted with EtOAc (3 x 2 mL), the combined organic layers were washed with brine ( ₃ mL), dried over _Na2SO4 , and concentrated under reduced pressure. Purification of the crude mixture by flash column chromatography (silica gel, eluent: 10:10:1 → 10:10:2 in hexane:EtOAc:MeOH) gave 17,18-unsaturated amidopyridine 47 (17 mg, 89% ). ¹ H NMR (500MHz, CDCl ₃ ) shift = 8.57 (d, J = 2.4Hz, 1H), 8.33 (dd, J = 1.2, 4.6Hz, 1H), 8.20 (d, J = 8.3Hz, 1H), 7.70 (s,1H),7.26(dd,J=4.9,7.8Hz,1H),6.55(br.s.,1H),5.77(s,1H),5.36(d,J=2.9Hz,1H),4.03 -3.89(m,4H),2.63-2.56(m,2H),2.52(ddd,J＝2.9,7.3,17.1Hz,1H),2.52-2.37(m,3H),2.36-2.27(m,2H) ,2.20(t,J=12.2Hz,1H),2.12(d,J=13.2Hz,1H),1.97(dd,J=2.2,12.9Hz,1H),1.89(td,J=8.8,13.2Hz, 1H), 1.78(tdd, J=2.4, 4.8, 12.8Hz, 1H), 1.71-1.62(m, 2H), 1.11(s, 3H). HRMS(ESI)(m/z) C ₂₇ H ₃₁ N ₂ O ₄ [M+H] ⁺ : calculated value 447.2278, measured value 447.2289.

Example S6. General method for the synthesis of ketones

To a solution of the ketal in THF was added 6N HCl (THF:6N HCl=1:1, 0.05M) at 0°C. The mixture was warmed to room temperature and stirred for 1 hour. The reaction was quenched with 6N NaOH and ethyl acetate and the layers were separated. The aqueous layer was extracted with ethyl acetate. The organic layers were combined, dried _{over Na2SO4} , and concentrated under reduced pressure.

Synthesis of Ketone 45

The crude mixture was purified by flash column chromatography (silica gel, eluent: 15:1 dichloromethane:MeOH) to afford ketone 45 (8.5 mg, 95%). ¹ H NMR (500MHz, CDCl ₃ ) shift=8.60(s,1H),8.52(d,J=3.9Hz,1H),7.74(br.s.,1H),7.28(dd,J=4.4,8.3Hz ,1H),5.91(s,1H),5.37(dd,J=2.7,4.6Hz,1H),3.88(d,J=13.7Hz,1H),3.84(d,J=13.7Hz,1H),2.91 (d,J=14.6Hz,1H),2.82(t,J=9.0Hz,1H),2.65(d,J=15.1Hz,1H),2.64(dd,J=10.3,14.6Hz,1H),2.59 -2.41(m,3H),2.32-2.20(m,3H),2.19-2.07(m,3H),1.85-1.72(m,2H),1.72-1.61(m,2H),1.44(br.s. ,1H),0.82(s,3H).HRMS(ESI)(m/z)C ₂₅ H ₃₁ N ₂ O ₂ [M+H] ⁺ : Calculated 391.2380, Found 391.2366.

Synthesis of Ketone 54

The crude mixture was purified by flash column chromatography (silica gel, eluent: 1:1 EtOAc:hexanes) to afford ketone 54 (8.7 mg, 80%). ¹ H NMR (500MHz, CDCl ₃ ) Shift = 9.54 (d, J = 10.0Hz, 2H) 8.04 (d, J = 10.0Hz, 1H), 7.93 (br.s., 2H), 6.40 (br.s. ,1H),5.95(br.s.,1H),5.47(br.m.,1H),2.95(d,J=10.0Hz,1H),2.77(m,1H),2.71-2.62(m,2H ),2.59-2.44(m,7H),2.34(t,J=10.0Hz,1H),2.19(t,J=10.0Hz,1H),2.00(m,1H),1.78(m,1H),1.18 (s,3H).HRMS(ESI)(m/z)C ₂₇ H ₂₇ N ₂ O ₂ [M+H] ⁺ : calculated value 411.5155, found value 411.5152.

Synthesis of Ketone 57

The crude mixture was purified by flash column chromatography (silica gel, eluent: 1:1 EtOAc:hexanes) to afford ketone 57 (13.7 mg, 72%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=8.04(br.s.,1H),7.68(d,J=10.0Hz,1H),7.48(br.s.,1H),7.27(d,J=10.0 Hz,1H),6.13(br.s.,1H),5.93(br.s.,1H),5.45(br.t.,J=5Hz,1H),2.97(d,J=15.0Hz,1H) ,2.76-2.64(m,3H),2.53-2.43(m,6H),2.40-2.34(m,2H),2.17(t,J＝10.0Hz,1H),1.98(m,1H),1.77(m ,1H),1.11(s,3H).HRMS(ESI)(m/z)C ₂₆ H ₂₇ N ₂ O ₂ [M+H] ⁺ : Calculated 399.5048, Found 399.5047.

Synthesis of Ketone 60

The crude mixture was purified by flash column chromatography (silica gel, eluent: 1:1 → 3:1 EtOAc:hexane, buffered with 2% triethylamine) to afford ketone 60 (3.3 mg, 68%). ¹ H NMR (500MHz, CDCl ₃ ) shift = 9.93-10.27 (br s, 1H), 8.06 (s, 1H), 7.75 (s, 1H), 7.47 (ABq, J _AB = 8.8Hz, Δν = 29.5Hz, 2H), 6.03(dd, J=2.0, 2.0Hz, 1H), 5.94(s, 1H), 5.47(dd, J=3.9, 3.9Hz, 1H), 2.97(d, J=15.1Hz, 1H), 2.63-2.76(m,3H),2.53-2.59(m,1H),2.44-2.50(m,5H),2.35-2.42(m,2H),2.17(dd,J＝9.3,9.3Hz,1H), 1.95-2.01(m,1H),1.74-1.80(m,1H),1.11(s,3H).HRMS(ESI)(m/z)C ₂₆ H ₂₇ N ₂ O ₂ [M+H] ⁺ : calculation Value 399.2073, found value 399.2043.

Synthesis of Ketone 22

See "Synthesis of Ketone 13" for this method. The resulting residue was then purified by flash chromatography (silica gel, eluent: 3:2→1:2 hexane:EtOAc) to afford ketone 22 (8.2 mg, 61%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.24(br.s.,1H),8.51(d,J=5.4Hz,1H),7.94(s,1H),7.84-7.76(m,2H),7.63 (d,J=5.4Hz,1H),6.27(br.s.,1H),5.97(s,1H),5.50(dd,J=2.4,4.9Hz,1H),2.98(d,J=14.6Hz ,1H),2.78(dd,J=6.8,11.2Hz,1H),2.71(d,J=14.6Hz,1H),2.72-2.63(m,1H),2.61(d,J=5.4Hz,1H) ,2.59-2.54(m,2H),2.54-2.50(m,2H),2.50-2.42(m,2H),2.39(ddd,J＝1.5,11.0,12.9Hz,1H),2.20(ddd,J＝ 1.5,9.5,11.5Hz,1H),2.01(ddd,J＝7.3,8.8,12.7Hz,1H),1.79(dt,J＝7.3,11.2Hz,1H),1.18(s,3H).HRMS(ESI )(m/z)C ₂₈ H ₂₈ NO ₂ [M+H] ⁺ : calculated value 410.2115, measured value 410.2111.

Synthesis of Ketone 48

See "Synthesis of Ketone 13" for this method. The resulting residue was then purified by flash chromatography (silica gel, eluent: 20:10:3 hexane:EtOAc:2M _NH3 in MeOH) to afford ketone 48 (5.0 mg, 74%). ¹ H NMR (500MHz, CDCl ₃ ) shift = 8.60 (d, J = 2.0Hz, 1H), 8.37 (dd, J = 1.0, 4.9Hz, 1H), 8.24-8.20 (m, 1H), 7.55 (s, 1H), 7.30(dd, J=4.9, 8.3Hz, 1H), 6.57(br.s., 1H), 5.94(s, 1H), 5.48(dd, J=2.2, 5.1Hz, 1H), 2.95( d,J=15.1Hz,1H),2.69(d,J=14.6Hz,1H),2.68(d,J=12.7Hz,1H),2.66-2.61(m,2H),2.61-2.53(m,2H ),2.52-2.44(m,3H),2.41(d,J=18.1Hz,1H),2.29(ddd,J=1.5,11.1,12.8Hz,1H),2.22-2.15(m,J=1.5,9.4 ,11.1Hz,1H),1.98(ddd,J＝7.6,9.0,12.7Hz,1H),1.76(dt,J＝7.3,11.2Hz,1H),1.14(s,3H).HRMS(ESI)(m /z) C ₂₅ H ₂₇ N ₂ O ₃ [M+H] ⁺ : calculated value 403.2016, found value 403.2023.

Example S7. General method for the synthesis of N-oxides

To a solution of the amine (1.00 equiv) in methanol ( _0.028M ) was added H2O2 ₍ 32.0 equiv) at room temperature. After 25 hours, saturated NaHCO ₃ solution was added, diluted with dichloromethane, and the layers were separated. The aqueous layer was extracted with dichloromethane. The organic layers were combined, washed with brine, dried _{over Na2SO4} and concentrated under reduced pressure.

Synthesis of 14B-N-oxide (14BNO)

The crude mixture was purified by flash column chromatography (silica gel, eluent: 90:9:1→80:18:2 in chloroform:methanol:5N _NH4OH in water) to give the N-oxide 14BNO (23.5 mg, 95 %). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.21(s,1H),8.48(d,J=5.9Hz,1H),7.77(s,1H),7.75(d,J=8.3Hz,1H),7.61 (d,J=5.9Hz,1H),7.57(dd,J=1.0,8.8Hz,1H),5.76(s,1H),5.28(d,J=2.9Hz,1H),3.44-3.36(m, 1H), 3.22(s, 3H), 3.12(t, J=10.8Hz, 1H), 3.10(d, J=1.0Hz, 3H), 2.47(dd, J=8.8, 11.2Hz, 1H), 2.44- 2.29(m,5H),2.28-2.13(m,4H),2.09(ddd,J=1.5,9.3,11.2Hz,1H),2.06-1.97(m,2H),1.94(dd,J=5.1,17.4 Hz,1H),1.85(dq,J=5.4,12.2Hz,1H),1.83-1.76(m,1H),1.72(td,J=9.3,12.2Hz,1H),0.54(s,3H).HRMS (ESI)(m/z) C ₃₀ H ₃₇ N ₂ O ₂ [M+H] ⁺ : Calculated 457.2850, Found 457.2842.

Synthesis of 14A-N-oxide (14ANO)

The crude mixture was purified by flash column chromatography (silica gel, eluent: 90:9:1→80:18:2 in chloroform:methanol:5N _NH4OH in _H20 ) to give the N-oxide 14ANO (3.6 mg, 77%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.80(s,1H),7.77(d,J=8.8Hz,1H),7.64 (d, J=5.9Hz, 1H), 7.60(d, J=8.8Hz, 1H), 5.81(s, 1H), 5.36(d, J=2.9Hz, 1H), 3.46(t, J=12.4Hz ,1H),3.24(s,3H),3.18(s,3H),3.16(t,J=9.8Hz,1H),2.64(dd,J=3.2,7.1Hz,1H),2.59-2.47(m, 3H), 2.43-2.28(m, 4H), 2.28-2.15(m, 2H), 2.09-1.99(m, 2H), 1.97(dd, J=5.1, 12.4Hz, 1H), 1.88(dq, J= 5.4,12.2Hz,1H),1.81-1.71(m,2H),1.51(dq,J=4.1,12.3Hz,1H),0.56(s,3H).HRMS(ESI)(m/z)C ₃₀ H ₃₇ N ₂ O ₂ [M+H] ⁺ : calculated value 457.2850, measured value 457.2846.

N-oxide formation of cortistatin A

Cortistatin A N-oxide: Aldrich silica gel Davisil ^™ (200 mesh) (200 mg) was added to a solution of cortistatin A (2 mg) in ethyl acetate (1 mL), and the solution was exposed to air and stirred for 1 hour. Silica gel was filtered and the filtrate was concentrated to give crude cortistatin A N-oxide, which was further purified by _SiO2 chromatography (eluent: 50% methanol/ethyl acetate) to give cortistatin A N-oxide ( 1.8 mg, 90% yield). ¹ H NMR (500MHz, CDCl ₃ ) δ9.22(s, 1H), 8.50(d, J=5.8Hz, 1H), 7.78(s, 1H), 7.76(d, J=8.8Hz, 1H), 7.63 (d,J=5.9Hz,1H),7.58(d,J=8.8Hz,1H),6.28(s,1H),5.49(m,1H),4.15(d,J=9.3Hz,1H),3.83 (t,J=9.8,9.8Hz,1H),3.31-3.36(m,1H),3.26(s,3H),3.19(s,3H),3.16(dd,J=9.3,9.3Hz,1H), 2.50(dd,J＝11.7,8.8Hz,1H),2.14-2.40(m,5H),1.97-2.07(m,3H),1.81-1.90(m,2H),1.68-1.75(m,1H), 1.49-1.65(m,1H),0.55(s,3H).HRMS(ESI)(m/z)C ₃₀ H ₃₇ N ₂ O ₄ [M+H] ⁺ : Calculated 489.2753, Found 489.5928.

Example S8. Synthesis of C3-alcohols and substituted analogs

Synthesis of β-alcohol 17B

β-alcohol 17B: A solution of ketone 13 (2.00 mg, 4.85 μmol, 1.0 equiv) in THF (350 mL) was cooled to −78 °C and a solution of L-selectride in THF (1 M, 9.71 μL, 9.71 mmol, 2.0 equivalent). After 1 h, saturated _NH4Cl solution (400 μL) and ethyl acetate (300 μL) were added and allowed to warm to room temperature. The aqueous phase was extracted with ethyl acetate (3 x 1 mL), the combined organic phases were washed with brine ( ₁ mL), dried over _Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: 1:1 hexane:EtOAc) to afford β-alcohol 17B (ca. 1.2 mg, 60%).

¹ H NMR (600MHz, CDCl ₃ ) shift = 9.26 (br.s, 1H), 8.49 (br.s, 1H), 7.82 (s, 1H), 7.78 (d, J = 8.2Hz, 1H), 7.69 ( br.s,1H),7.63(d,J=7.6Hz,1H),5.75(s,1H),5.26(br.s,1H),4.36(br.s,1H),3.15(t,J= 9.7Hz, 1H), 2.63(t, J=13.5Hz, 1H), 2.51(dd, J=9.1, 10.9Hz, 1H), 2.42-2.28(m, 2H), 2.24(t, J=10.6Hz, 1H), 2.21-2.12(m, 2H), 2.12-1.97(m, 3H), 1.93(dd, J=5.0, 17.3Hz, 2H), 1.90-1.80(m, 2H), 1.79-1.58(m, 3H),0.54(s,2H).HRMS(ESI)(m/z)C ₂₈ H ₃₂ NO ₂ [M+H] ⁺ : Calc. 414.2428, Found 414.2436.

α-ol 17A

A solution of ketone 13 (9.6 mg, 23.3 μmol, 1.00 equiv) in THF (750 mL) was cooled to −78° C., and a solution of LAH in ether (1.0 M, 35.0 L, 35.0 μmol, 1.50 equiv) was added. After 10 min, saturated _NH4Cl solution (500 μL) and ethyl acetate (500 μL) were added and allowed to warm to room temperature. The aqueous phase was extracted with ethyl acetate (3 x 1 mL), the combined organic phases were washed with brine ( ₁ mL), dried over _Na2SO4 and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, eluent: 1:1→1:5 in hexanes:EtOAc→100% EtOAc) to afford α-alcohol 17A (8.5 mg, 88%). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.22(s,1H),8.47(d,J=5.3Hz,1H),7.79(s,1H),7.75(d,J=8.2Hz,1H),7.63 (d,J=5.9Hz,1H),7.59(dd,J=1.2,8.8Hz,1H),5.75(s,1H),5.28(d,J=2.3Hz,1H),3.78(tt,J= 4.0,11.3Hz,1H),3.14(t,J=10.0Hz,1H),2.51(dd,J=8.5,11.4Hz,1H),2.40-2.33(m,2H),2.32(dt,J=4.7 ,12.3Hz,1H),2.28-1.98(m,7H),1.93(dd,J=5.0,17.3Hz,1H),1.90-1.81(m,2H),1.74-1.62(m,2H),1.40( dtd,J＝5.9,11.6,13.8Hz,1H),0.53(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₂ NO ₂ [M+H] ⁺ : calculated value 414.2428, measured value 414.2437 .

α-Methyl ether 64A

To a solution of α-alcohol 17A (2.0 mg, 4.83 mmol, 1.00 equiv) in DMF (300 mL) was added 60 wt% NaH (1.0 mg, 24.1 mmol, 5.00 equiv) at room temperature and pre-stirred for 30 minutes. The temperature was lowered to -10°C and MeI (2.0 μL, 29.0 mmol, 6.00 equiv) was added. After 2.5 hours, 2M NaOH solution (200 μL) and ethyl acetate (500 μL) were added and allowed to warm to room temperature. The aqueous phase was extracted with ethyl acetate (3 x 1 mL), the combined organic phases were washed with brine ( ₁ mL), dried over _Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: 1:2 hexane:EtOAc) to afford α-methyl ether 64A (ca. 1.2 mg, 58%). ¹ H NMR (600MHz, CDCl ₃ ) Shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.75(d,J=8.8Hz,1H),7.63 (d,J=5.9Hz,1H),7.59(dd,J=1.5,8.5Hz,1H),5.74(s,1H),5.28(d,J=2.9Hz,1H),3.39(s,3H) ,3.29(tt,J=3.5,11.4Hz,1H),3.14(t,J=10.0Hz,1H),2.52(dd,J=8.5,11.4Hz,1H),2.42-2.29(m,3H), 2.25(t, J=11.7Hz, 1H), 2.24-2.08(m, 5H), 2.06(td, J=4.1, 12.9Hz, 1H), 1.93(dd, J=5.3, 17.0Hz, 1H), 1.86 (dq,J=5.3,12.3Hz,1H),1.79(t,J=12.0Hz,1H),1.75-1.61(m,2H),1.32(dtd,J=4.7,11.5,14.0Hz,1H), 0.53(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₂ NO ₂ [M+H] ⁺ : Calculated 428.2584, Found 428.2573.

β-methyl ether 64B

The reaction conditions are the same as for the synthesis of α-methyl ether 64A. The residue was purified by preparative TLC (eluent: 1:2 hexane:EtOAc) to afford C3β-methyl ether 64B (1.2 mg, 58%). ¹ H NMR (500MHz, CDCl ₃ ) shift=9.22(s,1H),8.48(d,J=5.9Hz,1H),7.79(s,1H),7.76(d,J=8.8Hz,1H),7.63 (d,J=5.9Hz,1H),7.60(dd,J=1.5,8.3Hz,1H),5.74-5.70(m,1H),5.25(dd,J=2.2,5.1Hz,1H),3.75- 3.69(m,1H),3.35(s,3H),3.18-3.10(m,J=9.3Hz,1H),2.51(dd,J=8.5,11.5Hz,1H),2.52-2.44(m,1H) ,2.40-2.26(m,4H),2.17(s,3H),2.14-2.08(m,1H),2.07-1.96(m,2H),1.96-1.90(m,2H),1.86(dq,J= 4.9,12.0Hz,1H),1.76-1.61(m,1H),1.55-1.45(m,1H),0.54(s,3H).HRMS(ESI)(m/z)C ₂₈ H ₃₂ NO ₂ [M +H] ⁺ : calculated value 428.2584, measured value 428.2599.

α-Monomethylcarbamate 68A

To a solution of α-alcohol 17A (3.5 mg, 8.5 μmol, 1.00 equiv) in _CH3CN (350 μL) was added CDI (2.1 mg, 12.7 μmol, 1.50 equiv) and the reaction mixture was refluxed for 4 hours. The crude mixture was concentrated under reduced pressure and used in the next reaction without further purification.

To a solution of the crude mixture in DCM (300 mL) was added _MeNH2 in THF (2M, 50 μL) at room temperature and stirred for 14 h. The crude mixture was concentrated under reduced pressure and purified by prep-TLC (eluent: 1:1 hexane:EtOAc) to afford C3α-monomethylcarbamate 68A (2.4 mg, 60% for 2 steps). ¹ H NMR (500MHz, CDCl ₃ ) Shift=9.28(s,1H),8.50(d,J=5.9Hz,1H),7.87(s,1H),7.83(d,J=8.8Hz,1H),7.75 (d,J=5.9Hz,1H),7.69(d,J=8.8Hz,1H),5.77(s,1H),5.30(dd,J=2.2,4.6Hz,1H),4.76(t,J= 11.7Hz, 1H), 4.61(br.s., 1H), 3.17(t, J=10.0Hz, 1H), 2.82(d, J=4.4Hz, 3H), 2.53(dd, J=8.5, 11.5Hz ,1H),2.38(d,J=17.1Hz,2H),2.32(dd,J=3.7,11.5Hz,1H),2.30-2.14(m,5H),2.13-2.01(m,2H),1.95( dd,J=5.1,17.3Hz,1H),1.94-1.82(m,2H),1.78-1.67(m,2H),1.43(dq,J=4.9,12.7Hz,1H),0.55(s,3H) .HRMS(ESI)(m/z) C ₃₀ H ₃₅ N ₂ O ₃ [M+H] ⁺ : Calculated 471.2642, Found 471.2631.

α-Methoxyethyl ether 63A

To a solution of α-alcohol 17A (2.0 mg, 4.83 μmol, 1.00 eq) in DMF (300 mL) was added 60 wt% NaH (1.0 mg, 24.1 μmol, 5.00 eq) at room temperature and pre-stirred for 30 minutes, then MeO ( CH ₂ ) ₂ Br (1.6 μL, 16.6 μmol, 3.00 equiv). After 36 hours, 2M NaOH solution (200 μL) and ethyl acetate (500 μL) were added. The aqueous phase was extracted with ethyl acetate (3 x 1 mL), the combined organic phases were washed with brine ( ₁ mL), dried over _Na2SO4 and concentrated under reduced pressure. The residue was purified by preparative TLC (eluent: 2:5 hexane:EtOAc) to afford C3α-methoxyethyl ether 63A (ca. 0.7 mg, 27%). ¹ H NMR (600MHz, CDCl ₃ ) Shift=9.24(s,1H),8.50(d,J=5.9Hz,1H),7.81(s,1H),7.77(d,J=8.3Hz,1H),7.65 (d,J=5.9Hz,1H),7.61(d,J=8.8Hz,1H),5.75(s,1H),5.29(d,J=2.4Hz,1H),3.74-3.63(m,2H) ,3.61-3.51(m,2H),3.44(tt,J＝3.9,11.7Hz,1H),3.44-3.39(s,3H),3.16(t,J＝9.8Hz,1H),2.54(dd,J =8.5,11.5Hz,1H),2.43-2.30(m,3H),2.30-2.17(m,4H),2.17-2.02(m,3H),1.95(dd,J=5.1,17.3Hz,1H), 1.92-1.82(m,2H),1.78-1.62(m,J=8.3Hz,2H),1.41(dtd,J=4.1,11.9,13.5Hz,1H),0.55(s,3H).HRMS(ESI) (m/z)C ₂₈ H ₃₂ NO ₂ [M+H] ⁺ : calculated value 472.2846, found value 472.2850.

Example S9. Synthesis of amines from alcohols

α-Dimethylhydantoin 74A

To a solution of β-alcohol 17B (5.0 mg, 12.3 μmol, 1.0 equiv) in THF (400 μL) was added dimethylhydantoin (7.8 mg, 61.6 μmol, 5.0 equiv) and PPh ₍ 9.7 mg, 36.9 μmol, 3.0 equiv). The reaction mixture was cooled to 0° C., and DEAD (16.1 μL of 40 wt% in toluene, 36.9 mol, 3.0 equiv) was added. The reaction was warmed to 50 °C and stirred for 17 hours. After cooling the reaction mixture to room temperature, 1N NaOH solution (300 μL) was added and the aqueous phase was extracted with ethyl acetate (3×0.5 mL), the combined organic phases were washed with brine (1 mL), dried over Na ₂ SO ₄ , Concentrate under reduced pressure. The crude mixture was purified by preparative TLC (silica gel, eluent: 40:1 MeOH:dichloromethane) to afford α-dimethylhydantoin 74A (0.9 mg, 14%). ¹ HNMR (500MHz, CDCl ₃ ) shift=9.24(s, 1H), 8.50(d, J=5.4Hz, 1H), 7.81(s, 1H), 7.77(d, J=8.8Hz, 1H), 7.64( d,J=5.4Hz,1H),7.61(dd,J=1.5,8.8Hz,1H),5.79(d,J=1.5Hz,1H),5.32(dd,J=2.7,5.1Hz,1H), 5.18(s,1H),4.10(tdd,J=3.2,11.3,13.1Hz,1H),3.16(dd,J=9.3,10.7Hz,1H),2.86(t,J=12.7Hz,1H),2.54 (dd, J=8.3,11.7Hz,1H),2.45(dd,J=2.9,14.6Hz,1H),2.30(br.s.,6H),2.19(tq,J=4.4,9.0Hz,1H) ,2.09-2.00(m,1H),1.96(dd,J＝5.4,17.6Hz,1H),1.92-1.80(m,2H),1.80-1.64(m,3H),1.42(d,J＝4.9Hz ,6H),0.56(s,3H).HRMS(ESI)(m/z)C ₃₃ H ₃₈ N ₃ O ₃ [M+H] ⁺ : Calcd. 524.2908, Found 524.2892.

II. CDK8/19 Inhibitors

In any of the embodiments described herein, CDK8/19 inhibitors other than cortistatin may be used in combination with methods of targeting the selection of patients for treatment using the biomarkers identified herein. A series of CDK8/19 inhibitors are known in the art, including but not limited to those described in the following publication: Schiemann, K. et al. Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore. Bioorg. Med. Chem. Lett. 26, 1443 –1451 (2016); Mallinger, A. et al. Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19. J. Med. Chem. 59, 1078–1101 (2016); Koehler, M., Bergeron, P. & Blackwood, E.M. Potent, Specific CDK8Kinase Inhibitors Lack CDK8-dependent Antiproliferative Activity in HCT-116 Colon Cancer CellLine.ACSMed.Chem.Lett.(2016).doi:10.1021/acsmedchemlett.5b00278; . A selective chemical probe for exploring the role of CDK8 and CDK19 in humandisease. Nat. Chem. Biol. 11, 973–980 (2015).

The following US patent applications provide additional non-limiting examples of CDK8/19 inhibitors known in the art: US2013/0217014; US2015/027953; US2004/0180848; US2004/018844; US2014/0038958; 0229484；US2005/0009846；US2008/0287439；US2010/0093769；US2005/0256142；US2003/0018058；US2001/0047019；US2002/002178；US2009/0318441；US2005/0192300；US2009/0325983；US2006/0235034；US2010/0215644； US2010/00120781；US2006/0183760；US2009/0270427；US20020165259；US2006/0241297；US2004/0186288；US2006/0241112；US2006/0270687；US2006/0270687；US2004/0254094；US2003/0176699；US2006/0148824；US2003/0114672； US2009/0215805; US2009/0137572 and US2007/0161635.

In one embodiment, the CDK8/19 inhibitor is an analog of Senexin. In another embodiment, the CDK8/19 inhibitor is an analog of Selvita.

III. Targeted selection of patients suitable for CDK8/19 inhibitor therapy

Certain patients with tumors or cancers have been found to be more prone to respond to cortistatin therapy than others, and these patients can be identified by analyzing specific biomarkers in the patient's tumor or cancer, which will be described in detail below . Using methods known in the art in conjunction with the methods disclosed herein, a healthcare provider can determine whether a patient will respond successfully to cortistatin therapy. The present invention allows the identification of patients who would benefit from cortistatin therapy as candidates for treatment and is the basis for excluding patients who are unlikely to respond.

As used herein, the term "biomarker" refers to an indicator, such as predictive, diagnostic and/or prognostic, that can be detected in a sample. Biomarkers can serve as indicators of a particular subtype of a disease or disorder (eg, cancer) that is characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, a biomarker is a gene or a combination of genes. In some embodiments, a biomarker is a protein or combination of proteins. In other embodiments, the biomarkers are a combination of genes and proteins. In some embodiments, the biomarker is a protein expressed by the gene. Biomarkers include, but are not limited to, polynucleotides (eg, DNA and/or RNA), polypeptides, polypeptide and polynucleotide modifications (eg, post-translational modifications), carbohydrate and/or glycolipid-based molecular markers. In yet another embodiment, the biomarker is protein localization, such as RUNX1 enriched at certain loci to determine the likelihood of patient response.

A. Patient Selection Based on Impaired RUNX1 Pathway

(RUNX1) is a major hematopoietic transcription factor (TF) that regulates the differentiation of hematopoietic stem cells into mature blood cells. It is also sometimes called acute myeloid leukemia 1 protein (AML1) or core binding factor subunit alpha-2 (CBFA2). RUNX1 has been reported to regulate the differentiation of hematopoietic stem cells into mature blood cells, and more than 35 RUNX1 inactivating mutations have been identified to be implicated in various malignancies. Such inactivating mutations include, but are not limited to, RUNX1 point mutations, chromosomal translocations involving the RUNX1 gene, and mutations that result in increased destabilization or degradation of the RUNX1 protein. For an overview of exemplary RUNX1 inactivating mutations known to be associated with cancer, see, e.g., Ito et al., The RUNX family: developmental regulators cancer, Nature Reviews Cancer 15, 81–95 (2015), e.g. the last paragraph on page 83 to Page 84, last paragraph, Tables 1 and 2; and Ley et al., Genomic and Epigenomic Landscapes of Adult De NovoAcute Myeloid Leukemia, NEJM 368:22, 2059-74 (2013); the entire contents of which are incorporated herein by reference. Although knowledge about RUNX1 mutations in cancer has led to insights into the molecular pathology of various malignancies, inactivation of transcription factors such as RUNX1 has been difficult to treat or correct through clinical intervention.

Inactivating RUNX1 mutations have also been observed in non-hematopoietic malignancies such as breast cancer and can contribute to solid tumor formation (Ito et al., The RUNX family: developmental regulators in cancer, Nature Reviews Cancer 15, 81–95 (2015); Ellis, M.J. et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature 486, 353–360 (2012); Banerji, S. et al. Sequence analysis of mutations and translocations across breast cancer subtypes. Nature 486, 405–4209) (). Furthermore, RUNX1 downregulation is evident in solid tumor metastasis compared with primary tumors, and its reduced expression is part of a 17-gene signal associated with metastasis (Ramaswamy, S., Ross, K.N., Lander, E.S. & Golub, T.R.A molecular signature of metastasis in primary solid tumors. Nature Genet. 33, 49–54 (2003)).

Demonstration that inhibition of CDK8/19 activates the RUNX1 program includes: 1) Cortistatin A markedly increases the expression of a number of RUNX1 target genes, including CEBPA, IRF8, and NFE2, in AML cell lines including cells from the diagnosed myeloid MOLM-14 in patients with dysplastic syndromes that convert to AML; 2) cortistatin A induces RUNX1 recruitment to loci upregulated by cortistatin, suggesting that CDK8/19 kinase activity blocks RUNX1 activation at target loci accumulation; and 3) the antiproliferative activity of cortistatin was positively correlated with cell lines with impaired RUNX1 target gene expression, including those carrying RUNX1 mutations.

In general, mutations in the RUNX1 gene that result in impaired RUNX1 activity are associated with changes in the amino acid sequence of the RUNX1 protein compared to the wild-type (non-mutated) RUNX1 sequence. Such mutations leading to abnormal RUNX1 protein include, for example, substitutions, deletions or duplications of amino acids or amino acid sequences, frameshifts or premature stop codons in the protein coding sequence of RUNX1, or the fusion of RUNX1 protein sequences or fragments thereof with heterologous proteins or Fusion of fragments. This fusion is usually the result of a chromosomal translocation, resulting in the fusion of the genomic sequence encoding the RUNX1 protein or a fragment thereof with that encoding a different protein or fragment thereof.

The gene, transcript and protein sequences of these and other RUNX1 -binding partners or RUNX1 target genes are well known to those skilled in the art. Representative human RUNX1 binding partners and RUNX1 target genes are identified in Table 1 below.

One of ordinary skill in the art will be able to identify wild-type sequences of RUNX1 binding partners and RUNX1 target genes based on the identifiers provided above.

In some embodiments, the cancer comprises a RUNX1-RUNX1T1 translocation. The RUNX1-RUNX1T1 translocation is well known in the art. See eg Kim et al., Acute myeloid leukemia with a RUNX1-RUNX1T1t(1;21;8)(q21;q22;q22) novel variant: a case report and review of the literature. Acta Haematol. 125(4):237- 41 (2011), the entire contents of which are incorporated herein by reference. Additional RUNX1 translocations associated with cancer are also known to those skilled in the art, such as RUNX1-ETO ETV6-RUNX1 and RUNX1-EVI1 translocations.

在一些实施方案中，癌症在由癌症基因组编码的RUNX1蛋白中包含A142_A149dup、A142fsX170、A149fsX、A251fsX、A338fsX482、A63fsX、D160Y、D326fsX481、E223fsX、E422fsX、F411fsX482、G165R、G170fsX201、G394_L406dup、G394fsX482、G409fsX482、G439fsX482 、H105_F116dup、H105fsX541、H427fsX、I114fsX117、I342fsX、K215fsX269、L112fsX117、L144fsX170、L210fsX269、L313fsX323、L382fsX482、L98fsX、N448_V452dup、P113A、P345R、P464P、P95fsX117、Q335_L339dup、Q438fsX482、R107C、R107S、R166Q、R201G、R201Q、R201X , R232W, R320X, R346fsX, R346fsX482, S141fsX, S226fsX269, S256fsX269, S322fsX323, S331fsX, S388fsX481 , T148fsX170, Y355fsX, or Y380fsX482 mutations, or any combination thereof. According to the accession number NC_000021.8 (36160098..36421595, complementary sequences) of the NCBI compilation GRC37.p13 (GCF_000001405.25), annotation version 105 (accessible at the National Center for Biotechnology Information (NCBI) website ncbi.org), the The mutation positions listed above and the mutation positions listed elsewhere herein for human RUNX1. Those skilled in the art will be able to identify homologous mutations in non-human individuals, for example by aligning human and non-human RUNX1 sequences and identifying corresponding residues, as is routinely done in the art. Furthermore, based on the sequence alignment and identification of homologous residues, one skilled in the art will be able to access any new annotated version of the NCBI database (e.g. NCBI Compendium GRCh38.p2 (GCF_000001405.28), accession number NC_000021. 9 (34787801..35049310, complementary)) where the mutations listed above were identified.

It has been discovered that cortistatin, or a pharmaceutically acceptable salt, quaternary ammonium salt or N-oxide thereof, as a CDK8/19 inhibitor can be used to counteract RUNX1 impairment and treat RUNX1 mutated cancers and binding partners or RUNX1 target genes therein Mutated cancers. CDK8 and CDK19 are sometimes referred to as "mediator kinases" because they are assembled in multiprotein complexes that bind the mediator complex in a reversible manner. The Mediator complex links enhancer-bound transcription factors to the promoter-bound RNA polymerase II holoenzyme and influences chromatin structure to regulate transcription and gene expression through as yet unclear mechanisms. Recent comprehensive whole-genome sequencing of samples from 200 AML patients revealed that nearly all mutations in putative cancer drivers are associated with regulation of gene expression. See, eg, Aerts, et al., Nature (2013) 499:35-36; The Cancer Genome Atlas Research Network, 2013. Genomic and Epigenomic Landscapes of Adult De NovoAcute Myeloid Leukemia. N. Engl. J. Med. 368, 2059-2074. Thus, aspects of the present disclosure are based on the recognition that specific inhibition of Mediator kinases, particularly CDK8 and CDK19, constitutes a novel means of disrupting the downstream effects of impaired RUNX1 activity in various cancers, particularly in blood in cancers such as AML.

In one example, in AML, RUNX1 regulates transcription along with other transcription factors and binding partners that may have mutations, including CBFb, GATA1/2, PU.1 and ERG. Impairment of RUNX1 affects this pathway. Furthermore, other mutations in AML may suppress the RUNX1 transcriptional program through RUNX1 protein degradation (MLL fusions) or genetic repression through DNA methylation (IDH2 mutations). Thus, targeting selection of RUNX1-impaired patients with cortistatin therapy represents a new broadly useful mechanism for activating the RUNX1 transcriptional program and thereby restoring more normal hematopoiesis, or making cells more normal and less toxic. Weak or induced maturation with potential for growth arrest and/or apoptosis.

In one aspect of the second embodiment, the biomarker is directly or indirectly related to the RUNX1 pathway. For example, to provide a method to determine whether a patient with a tumor or cancer can be successfully treated with cortistatin begins by assessing whether the patient carries an inactivating mutation of the RUNX1 gene or a gene involved in RUNX1-mediated transcription such as but not limited to Inactivating mutations of GATA1, GATA2, C/EBPa, FLI1, FOG1, ETS1, PU.1, ERG, and CBFα). RUNX1 inhibition (partial or complete) can be manifested by monoallelic inactivating mutations or translocation to RUNX1-RUNX1T1 (also known as AML1-ETO), which blocks wild-type RUNX1 DNA association and transcription.

In some embodiments, the diagnostic or therapeutic methods provided herein comprise detecting the expression level of RUNX1 of RUNX1, a RUNX1 binding partner, and/or a RUNX1 target gene, and comparing it to a reference level to determine whether a cancer exhibits impaired RUNX1 activity, where RUNX1 target genes are ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPα, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10 , E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4 , IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK , PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3 , ZBTB16 and ZCCHC5 or a combination thereof. In some embodiments, the RUNX1 target gene is BCL2, CCNA1, CD44, C/EBPa, CBFβ, CSF1, CXCL10, CXCR4, ETS1, ETS2, FLI1, FOG1, FCER1A, GATA1, GATA2, GFI1B, HEB, IRF1, IRF8, One or a combination of JAG1, LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1 and TNF.

In certain embodiments, the RUNX1 impaired tumor or cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myeloid leukemia, B cell Acute lymphoblastic leukemia (B-ALL), childhood B-ALL, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS Associated lymphoma, chronic myeloproliferative disorder, primary central nervous system lymphoma, T-cell lymphoma, hair cell leukemia, or multiple myeloma (MM).

In another embodiment, patients diagnosed with myelodysplastic syndrome (MDS) may be treated using the present invention. Many of the recurrent somatic mutations that drive the MDS phenotype reside in transcription factors and epigenetic targets that regulate transcription. RUNX1 is a transcription factor and master regulator of hematopoietic cells mutated in 10-20% of MDS patients, making it the most frequently mutated gene in MDS. Mutations in RUNX1 attenuate expression of target genes that drive differentiation, and this effect predicts a higher risk and shorter time to secondary AML (sAML) conversion. Inhibition of CDK8/19 has been found to increase the expression of RUNX1-target genes, and thus the present invention may be an effective therapeutic approach for treating MDS patients with RUNX1 mutations and other mutations that inhibit this key differentiation program.

Impaired RUNX1 activity, eg, due to loss-of-function mutations of the RUNX1 gene, is known to be associated with various forms of cancer including, for example, various types of leukemia. Since RUNX1 is an activating transcription factor and no strategies are available to compensate for the loss of transcriptional activation mediated by RUNX1, there are no clinical interventions to counteract impaired RUNX1 activity. Some aspects of the disclosure are based on the identification of a panel of RUNX1 binding partners and target genes. Aspects of the present disclosure are based on the recognition that the expression or activity of RUNX1 binding partners and target genes is modulated by administering certain compounds such as CDK8/19 inhibitors and/or cortistatin or cortistatin analogs thereof, These compounds alone or in combination with other compounds provided herein constitute an effective strategy for counteracting impaired RUNX1 activity, eg, for the treatment of individuals carrying cancers exhibiting impaired RUNX1 activity.

Accordingly, the present disclosure provides methods, compositions and kits for treating cancers exhibiting impaired RUNX1 activity. In addition, the present disclosure also provides methods for determining whether a cancer in an individual is sensitive to treatment with the compounds and compositions provided herein, and for selecting an agent for treatment based on such determination according to any of the methods of treatment and strategies provided herein. patient approach.

Those skilled in the art will appreciate that the present disclosure is not limited to RUNX1 mutations that result in impaired RUNX1 activity. For a review of RUNX1 mutations that result in impaired RUNX1 activity, see eg Ito et al., The RUNX family: developmental regulators in cancer, Nature Reviews Cancer 15, 81–95 (2015), eg last paragraph on pages 83 to 84 Last paragraph and Tables 1 and 2; Ley et al., Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia, NEJM 368:22, 2059-74 (2013); Gelsi-Boyer et al., Genome profiling of chronic myelomonocytic leukemia: frequent alterations of RAS and RUNX1genes. BMC Cancer 8, 299 (2008); and Kuo et al., RUNX1 mutations are frequent in chronic myelomonocytic leukemia and mutations at the C-terminal region can predict acute myeloid leukemia transformation. Leukemia 23, 1426- per The entire content of one is hereby incorporated by reference.

B. Select patients based on biomarkers other than RUNX

In another embodiment of the invention, a method for predicting the response of a patient with a tumor or cancer to cortistatin therapy comprises the steps of: obtaining a tumor or cancer sample from the patient; detecting one of the following in the biological sample from the patient; or the expression level or amount of multiple biomarkers, wherein the biomarkers are selected from the group consisting of: ER positive, VHL loss-of-function mutation (VHL negative), HER2 overexpression, EGFR mutation, MET mutation, neural Biomarkers for blastoma; inactivating mutations in EWS-FLI1, STAT1-pS727, STAT1, or ETV1, FLI1, SMC3, SMC1A, RAD21, or STAG2; determine whether the expression level or amount is higher or lower than in the corresponding The level or amount of expression found in normal cells, for example above or below a specific amount associated with an increase or decrease in clinical benefit to the patient; and then optionally with an effective amount of a CDK8/19 inhibitor or a pharmaceutically acceptable The patient is treated with the accepted salt, oxide, or pharmaceutically acceptable salt thereof. In another embodiment, the observed gene expression is compared to the expression of the same gene in a control sample comprising a representative number of patients who responded to a CDK8/19 inhibitor or a predictive animal model and a representative number of patients with no or poor response to CDK8/19 inhibitors to determine whether patients tend to respond to cortistatin therapy. If a patient's biomarkers indicate so, then healthcare providers can assume that the patient is more likely to respond to treatment.

In one embodiment, the neuroblastoma is also sensitive to a CDK8/19 inhibitor due to activation of the RUNX1 transcriptional program. See Inoue, K.-I. & Ito, Y. Neuroblastoma cell proliferation is sensitive to changes in levels of RUNX1 and RUNX3 protein. Gene 487, 151-155 (2011).

Examples of tumors and cancers with abnormal STAT1 or STAT1-pS727 levels are included in Timofeeva, O.A. et al. Serine-phosphorylated STAT1 is a prosurvival factor in Wilms' tumorogenesis. Oncogene 25, 7555-7564 (2006); ,L.&Wu,R.Differentialexpression of STAT1and IFN-γin primary and invasive or metastatic wilmstumors.J.Surg.Oncol.108,152–156(2013);Arzt,L.,Kothmaier,H.,Halbwedl,I.,Quehenberger, F. & Popper, H.H. Signal transducer and activator of transcription 1 (STAT1) acts like an oncogene in malignant pleural mesothelioma. Virchows Arch 465, 79–88 (2014). In one embodiment, the tumor or cancer associated with STAT1 or the STAT1-pS727 biomarker is mesothelioma and metastatic Wilms tumor.

IV. Diagnostics and Kits

Methods of obtaining cell or tissue samples from individuals containing cancer or tumor cells are well known to those of skill in the art. Such methods generally involve obtaining a tumor biopsy from the individual, eg, a tissue biopsy comprising cancer cells from a solid tumor or a bodily fluid biopsy comprising tumor cells from a liquid tumor. A person skilled in the art will know a suitable source of cancer cells in an individual depending on the type of cancer carried by the individual. For example, in some embodiments, the cancer is leukemia and the cancer cells are bone marrow cells, peripheral blood cells, or hematopoietic stem cells. In some such embodiments, the method comprises obtaining a blood or bone marrow sample from the individual comprising leukemia cells.

In one embodiment, the diagnostic methods provided herein determine whether a cancer in an individual is associated with or exhibits impaired RUNX1 activity. This impaired RUNX1 activity can be detected in different ways. For example, in some embodiments, impaired RUNX1 activity is detected by detecting mutations in the RUNX1 gene that have been reported to be associated with impaired RUNX1 activity. In some embodiments, by measuring the expression level of the RUNX1 gene product, such as the RUNX1 transcript, mRNA or protein level, in a cancer cell obtained from an individual, and comparing the measured level to that measured in a healthy cell of the same or similar cell type or expected reference levels to detect impaired RUNX1 activity. In some embodiments, if the measured expression level of RUNX1 in cancer cells is reduced by more than 25%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, compared to the level of RUNX1 expression in healthy cells, Impaired RUNX1 activity is detected by more than 80%, more than 90%, more than 95% or more than 98%. In the absence of a statement to the contrary, impaired RUNX1 activity is detected if the level of RUNX1 expression measured in cancer cells is reduced by more than 25% compared to the level of RUNX1 expression in healthy cells.

In some embodiments, the diagnostic methods provided herein comprise determining whether a cancer in an individual is associated with or exhibits impaired RUNX1 activity by detecting mutations in the cancer genome that result in impaired RUNX1 activity. A number of mutations leading to impaired RUNX1 activity have been reported previously. Such mutations include, but are not limited to, Gaidzik et al., RUNX1 mutations in acute myeloid leukemia: results from a comprehensive genetic and clinical analysis from the AML study group. J Clin Oncol. 29(10): 1364-72 (2011) Appendix Table A2 Those mutations that are disclosed; the entire contents of which are incorporated herein by reference. In some embodiments, the method comprises detecting a mutation in a gene encoding a RUNX1 protein, such as a mutation in the RUNX1 gene. In some embodiments, the method comprises detecting a mutation that results in a change in the amino acid sequence of the RUNX1 protein compared to the wild-type RUNX1 sequence. In some embodiments, the method comprises detecting mutations that result in substitutions, deletions, or duplications of amino acids or amino acid sequences, frameshifts, or premature stop codons in the protein coding sequence of RUNX1. In some embodiments, the mutation is a translocation resulting in an abnormal RUNX1 protein. In some embodiments, the mutation results in the deletion of a fragment of the RUNX1 protein. In some embodiments, the mutation results in the fusion of a genomic sequence encoding a RUNX1 protein or fragment thereof with a genomic sequence encoding a different protein or fragment thereof. In some embodiments, the mutation results in the fusion of a genomic sequence encoding a RUNX1 target protein or fragment thereof with a genomic sequence encoding a different protein or fragment thereof. In some embodiments, the mutation is a RUNX1-RUNX1T1 translocation.在一些实施方案中，突变是A142_A149dup、A142fsX170、A149fsX、A251fsX、A338fsX482、A63fsX、D160Y、D326fsX481、E223fsX、E422fsX、F411fsX482、G165R、G170fsX201、G394_L406dup、G394fsX482、G409fsX482、G439fsX482、H105_F116dup、H105fsX541、H427fsX、I114fsX117、 I342fsX、K215fsX269、L112fsX117、L144fsX170、L210fsX269、L313fsX323、L382fsX482、L98fsX、N448_V452dup、P113A、P345R、P464P、P95fsX117、Q335_L339dup、Q438fsX482、R107C、R107S、R166Q、R201G、R201Q、R201X、R232W、R320X、R346fsX、R346fsX482、 S141fsX, S226fsX269, S256fsX269, S322fsX323, S331fsX, S388fsX481, T148fsX170, Y355fsX, or Y380fsX482 mutations, or any combination thereof. The position of the mutation was defined according to the accession number cDNANC_000021.8 (accessible at the National Center for Biotechnology Information (NCBI) website at ncbi.org). Additional mutations that result in impaired RUNX1 activity will be apparent to those of skill in the art based on this disclosure and knowledge in the art. The present disclosure is not limited in this respect.

In some embodiments, the diagnostic methods provided herein comprise determining whether a cancer in an individual is associated with or exhibits impaired RUNX1 activity by detecting mutations in a gene encoding a RUNX1 binding partner or a RUNX1 target gene. For example, in some embodiments, the gene encoding a RUNX1 binding partner or the RUNX1 target gene is C/EBPa, CBFβ, ETS1, FLI1, FOG1, GATA1, GATA2, PU.1, TAL1, LMO2, or HEB. In some embodiments, the method comprises determining whether the cancer in the individual is associated with impaired RUNX1 activity or exhibits impaired RUNX1 activity by detecting MLL-AF9 translocations, MLL-AF4 translocations, Bcr-Abl fusions, or JAK2V617F mutations in the cancer. Impaired RUNX1 activity.

In some embodiments, the method comprises detecting the expression level of RUNX1, a RUNX1 binding partner, and/or a RUNX1 target gene, and comparing it to a reference level to determine whether the cancer exhibits impaired RUNX1 activity. In some embodiments, the RUNX1 target gene is selected from the group consisting of ACSL1, ADORA2B, ADRB1, AMPD3, ARRDC4, BCL2, BCL2A1, CBFβ, CCNA1, CD244, CD44, CDC42EP3, C/EBPa, CECR6, CFLAR, CISH, CSF1, CXCL10, CXCR4, CYTIP, DUSP10, E2F8, EMB, EMR2, ETS1, ETS2, FAM107B, FAM46A, FCER1A, FCGR1B, FLI1, FOG1, FOSL2, GAB2, GAS7, GATA1, GATA2, GFI1B, GMPR, GPR18, GPR183, HBBP1, HEB, HLX, HMGCS1, IGFBP4, IGFBP5, IL17RA, IL1RAP, IPCEF1, IRF1, IRF8, ITGA6, JAG1, LCP2, LDLR, LIMA1, LMO2, LRRC33, LTB, MBP, MICAL2, MYCN, MYO1G, NFE2, NOTCH2, NRP1, P2RY2, PAG1, PLAC8, PLEK, PLXNC1, PMP22, PTPRE, PU.1, PXK, RAB27A, RASA3, RGS16, RHOH, RNF24, RXRA, SELPLG, SLA, SLC7A11, SLC7A5, SOCS1, ST3GAL4, STK17B, TAL1, TIMP3, TMEM104, TNF, TSC22D1, TSC22D3, ZBTB16, and ZCCHC5. In some embodiments, the RUNX1 target gene is selected from the group consisting of: BCL2, CCNA1, CD44, C/EBPa, CBFβ, CSF1, CXCL10, CXCR4, ETS1, ETS2, FLI1, FOG1, FCER1A, GATA1, GATA2, GFI1B, HEB, IRF1, IRF8, JAG1, LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1, and TNF.

In some embodiments, the cancer comprises a MLL-AF9 translocation, a MLL-AF4 translocation, a Bcr-Abl fusion, or a JAK2V617F mutation. For a review of these translocations see, eg, Horton et al., MLL-AF9-mediated dimmortalization of human hematopoietic cells along different lineages changes during ontogeny. Leukemia 27(5):1116-26 (2013); Bueno et al., Insights into the cellular origin and etiology of the infant pro-B acute lymphoblastic leukemia with MLL-AF4 rearrangement. Leukemia. 25(3):400-10(2011); Press et al., BCR-ABL1RT-qPCR for monitoring the molecular response to tyrosine kinase inhibitors in chronic myeloid leukemia.J Mol Diagn.15(5):565-76(2013); and Mata et al., JAK2as a molecular marker in myeloproliferative diseases.CardiovascHematol Agents Med Chem.5(3):198-203(2007), which The entire contents are incorporated herein by reference.

In some embodiments, detecting impaired RUNX1 activity or impaired activity of a RUNX1 binding partner or a RUNX1 target gene comprises obtaining information about the presence or absence of one or more mutations in the RUNX1 gene, a gene encoding a RUNX1 binding partner, or a target gene and/or Or information about an increase or decrease in the expression level of a gene product encoded by such a gene. In some embodiments, such methods can include obtaining cancer cells from an individual to assess the genomic or expression status of RUNX1, a RUNX1 binding partner, or a RUNX1 target gene. In some embodiments, such methods include obtaining a biopsy of tissue or body fluid from an individual containing cancer cells, such as a tumor biopsy or a blood or bone marrow biopsy. In some embodiments, the cancer cells contained in the biopsy sample are then subjected to an assay suitable for detecting mutations or gene expression levels of RUNX1, RUNX1 binding partners, or RUNX1 target genes. In some embodiments, the biopsy may include normal cells or cells of an unwanted tissue type. For example, a peripheral blood or bone marrow sample may include cells that are not normally involved in the pathology of hematological cancers such as leukemia. In some embodiments, a biopsy sample is processed to enrich for cancer cells or cells often associated with cancer pathology (eg, hematopoietic stem cells), or to deplete cells not normally involved in carcinogenesis, such as differentiated cells. Methods of enriching and depleting cells from a sample obtained from an individual are well known to those skilled in the art.

In other embodiments, a biopsy sample is subjected to a detection assay without enrichment or depletion of specific cells or cell types.

One of ordinary skill in the art will be able to detect mutations and/or expression levels of RUNX1 or RUNX1 binding partners or RUNX1 target genes in biopsy samples from individuals by performing suitable assays known in the art on these samples. To detect mutations in coding genes, such assays typically involve obtaining genome sequence information from cancer cells. In some embodiments, the detection method comprises an assay comprising amplifying a target sequence, such as a genomic sequence encoding RUNX1 or a RUNX1 binding partner or a RUNX1 target gene, sequencing the amplified target sequence, and combining the sequence information obtained with The wild-type sequence is compared to determine if one or more mutations are present.

As used herein, the term "expression level" refers to information about the level of one or more gene products (eg, mRNA, protein, or combinations thereof) in a cell or tissue. In some embodiments, the detection of one or more gene mutations and/or the reduction of expression levels as described herein may be based on one or more measurements or assays, such as quantitative or semi-quantitative values of single gene expression, e.g. , reflecting a signal obtained from a quantitative or semi-quantitative assay detecting the abundance of a gene product (eg, a protein or nucleic acid transcript encoded by a RUNX1 gene, a RUNX1 binding partner, or a RUNX1 target gene). Suitable assays for detection of gene expression products are well known to those skilled in the art and include, for example, western blot, ELISA, RT-PCR (e.g., endpoint RT-PCR, real-time PCR, or qPCR), protein or nucleic acid microarrays, and large-scale Parallel sequencing analysis. However, any suitable assay based on hybridization, specific binding (eg, antibody binding), or any other technique may be used, as aspects of the invention are not limited herein.

In some embodiments, the presence and/or reduction in expression level of one or more genetic mutations as described herein may relate to multiple data points, such as quantitative or semi-quantitative values of expression and/or sequence or mutation data points. In some embodiments, the presence of one or more genetic mutations and/or increased or decreased expression levels described herein can be assessed in a biopsy sample. Methods described herein for detecting or generating data and/or increased or decreased expression levels of one or more gene mutations are well known to those skilled in the art and include, for example, Southern blots, western blots, ELISA, Northern blots, reverse Northern blot, RT-PCR (e.g. endpoint RT-PCR, real-time PCR or qPCR), PCR, ddPCR (e.g. droplet digital PCR), microarray (for protein or transcript detection), SNP analysis, PCR, hybridization analysis, Sequence analysis, etc. or any combination thereof (for exemplary detection methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition (3Volume Set), Cold Spring Harbor Laboratory Press; 3rd edition (January 15, 2001), ISBN -10:0879695773; Robert Grützmann (Editor), Christian Pilarsky (Editor), Cancer Gene Profiling: Methods and Protocols (Methods in Molecular Biology), Humana Press; 1st edition (November 6, 2009), ISBN-10:1934115762, both Both are incorporated herein by reference for disclosing definitive gene product detection and expression analysis methods).

In some embodiments, quantitative expression values are values that reflect the abundance of gene transcripts in a starting sample (eg, a tumor cell or tissue sample). In some embodiments, a semi-quantitative expression value is a value that reflects the abundance of a gene transcript in a starting sample relative to a control or reference amount, such as in a healthy cell or a cell of the same type obtained from a healthy individual Measured or expected quantity. Methods for calculating semi-quantitative expression values are well known to those skilled in the art. Suitable controls or reference amounts for generating semi-quantitative expression values are well known to those skilled in the art and include, for example, expression values of housekeeping genes such as β-actin or GAPDH, external controls such as spikes typically RNA or DNA controls that are not expressed in the cells of the cell), an overall expression value (such as all expression values obtained from the cells summed together), or a historical or empirical value.

In some embodiments, the expression level of RUNX1, a RUNX1 binding partner, or a RUNX1 target gene (eg, RNA and/or protein) determined for a sample is compared to a reference expression level. In some embodiments, a reference is a standard indicative of normal expression levels. In some embodiments, the reference is a standard indicative of a defective expression level (any test level at or below the reference would indicate impaired activity of RUNX1, a RUNX1 binding partner, or a RUNX1 target gene). In some embodiments, the reference level is obtained by determining the expression level of RUNX1, a RUNX1 binding partner, or a RUNX1 target gene in normal or healthy tissue. In some embodiments, the reference level is determined by assaying RUNX1, a RUNX1 binding partner, or a RUNX1 target gene in a reference sample obtained from the same individual from whom the test sample was obtained (eg, a sample that does not contain malignant cells). A reference sample can be obtained as a test sample from a different region of the same tissue or from a different region of the individual's body.

In some embodiments, an individual or a biopsy or other biological sample obtained from an individual is assessed to determine whether there is RUNX1 activity or impairment of the activity of a RUNX1 binding partner or a RUNX1 target gene, e.g., detected as a gene encoding RUNX1 , a mutation (eg, deletion, loss-of-function, frameshift, inversion, translocation, or other mutation) of a RUNX1 binding partner or a RUNX1 target gene, or a decreased expression level of RUNX1, a RUNX1 binding partner, or a RUNX1 target gene. It should be understood that any of the genetic and/or expressed information described herein may be used alone or in combination, with or without additional patient information, to aid in prognosis, treatment recommendations, or other diagnostic or predictive assessments of health, outcomes, and/or treatment of patients.

V. Methods and Pharmaceutical Compositions

The invention involves first assessing a patient in need of tumor or cancer treatment by determining whether the patient has abnormal levels of the biomarkers specified herein, and then, if the results warrant, treating the patient with cortistatin or CDK8/19 inhibitor therapy.

Cortistatin or other CDK8/19 inhibitors may be administered as a neat chemical, but more typically will be administered as a pharmaceutical composition comprising a selected corticostatin in an amount effective to a host (usually a human) in need of such treatment. or CDK8/19 inhibitors, as described herein. Accordingly, the present disclosure provides a pharmaceutical composition comprising an effective amount of cortistatin or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier for all uses described herein. The pharmaceutical composition may contain cortistatin as the sole active agent, or in an alternative embodiment, the pharmaceutical composition may contain the compound and at least one other active agent. In certain embodiments, the pharmaceutical composition is a pharmaceutical composition containing from about 0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about 5 mg to about 200 mg, or from about 5 mg to about 100 mg of the active compound and optionally other active agents in suitable doses. dosage form. Examples are dosage forms with at least 5, 10, 15, 25, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700 or 750 mg of active compound. Cortistatin or CDK8/19 inhibitors may be administered orally, topically, parenterally, by inhalation or spray, sublingually, via implants including ocular implants, transdermally, in dosage unit formulations containing conventional pharmaceutically acceptable carriers. Buccal administration, rectal, as an ophthalmic solution, injection, intravenous, intraaortic, intracranial, subcutaneous, intraperitoneal, subcutaneous, nasal, sublingual, or rectal or by other means.

In some embodiments, the present disclosure provides a compound comprising cortistatin or a CDK8/19 inhibitor such as formula (A-1), (A-1′), (A-1″), (A-2′), ( A-2″), (A-3′), (A-3″), (D1′), (D1″), (D2′), (D2″), (E1′), (E1″), (E2′), (E2″), (G1′) or (G1″) compounds or compositions of pharmaceutically acceptable salts, quaternary ammonium salts or N-oxides thereof, for administration to patients with Individuals with cancer or tumors with impaired RUNX1 activity. In some embodiments, the composition comprises a CDK8/19 inhibitor. In some embodiments, the composition further comprises a Jak1/2 inhibitor.

Although the description of pharmaceutical compositions provided herein primarily refers to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to animals as well. Modifications, if necessary, of pharmaceutical compositions suitable for administration to humans in order to adapt the compositions for administration to various animals are well understood and can be devised if necessary by those of ordinary skill in the art using only ordinary experimentation. and implement such changes. Individuals to whom the pharmaceutical composition is intended to be administered include, but are not limited to, humans and/or other primates; mammals such as cattle, pigs, horses, sheep, cats, dogs, rodents, mice, hamsters, and/or rats; birds; species such as chickens, ducks, geese and turkeys.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association the active ingredient with excipients and/or one or more other accessory ingredients, and then, if necessary and/or desired, shaping and/or packaging the product into desired unit doses. or multiple dosage units.

The pharmaceutical compositions according to the invention may be prepared, packaged and/or sold in bulk as a single unit dose and/or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete quantity of pharmaceutical composition containing a predetermined quantity of active ingredient. The amount of active ingredient is usually equal to the dose of active ingredient to be administered to the individual and/or a convenient fraction of that dose, for example one-half or a third of that dose.

Salts can be composed of inorganic acids sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chlorine Compounds, bromides, iodides such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, etc. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, lauryl salt, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthalene methanesulfonate , Glucoheptonate, Lactobionate, Lauryl Sulfonate and Isethionate etc. Salts can also be prepared from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like . Representative salts include acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, Maleate, Mandelate, Benzoate, Chlorobenzoate, Methylbenzoate, Dinitrobenzoate, Phthalate, Benzenesulfonate, Toluenesulfonic Acid Salt, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, etc. Pharmaceutically acceptable salts may include cations based on alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium, etc. as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, Tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. Salts of amino acids, such as arginine salts, gluconate salts, galacturonate salts, and the like are also contemplated. See, eg, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which is incorporated herein by reference.

The relative amounts of active ingredients, pharmaceutically acceptable excipients and/or any other ingredients in a pharmaceutical composition according to the invention will vary according to the identity, size and/or condition of the individual to be treated, and further depend on the combination The route by which the drug will be administered. For example, the composition may comprise from 0.1% to 100% (w/w) of an active ingredient such as a CDK8/19 inhibitor or cortistatin or a cortistatin analogue thereof, such as formula (A-1), (A -1′), (A-1″), (A-2′), (A-2″), (A-3′), (A-3″), (D1′), (D1″), (D2'), (D2"), (E1'), (E1"), (E2'), (E2"), (G1') or (G1") compound or a pharmaceutically acceptable salt thereof, Quaternary ammonium salts or N-oxides, and optionally any other active ingredients such as JAK1/2 inhibitors. In some embodiments, the composition comprises 0.1% to 1%, 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60% , 60% to 70%, 70% to 80%, 80% to 90% or 90% to 100% (w/w) active ingredient, more typically 0.1% to 100% (w/w) active ingredient.

The pharmaceutical formulations provided herein may additionally comprise a pharmaceutically acceptable excipient, as used herein, suitable for the particular dosage form desired, including any and all solvents, dispersion media, diluents or other liquid media, dispersion or suspension Auxiliaries, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and methods for preparing them. known technology. Except for any conventional excipient medium which is incompatible with the substance or its derivatives, for example by producing any undesired biological effect or interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is considered is within the scope of the present invention.

In some embodiments, the excipient is one that has been approved for human and veterinary use, eg, by the US Food and Drug Administration. In some embodiments, the excipient complies with the standards of the United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia.

Pharmaceutically acceptable excipients for the preparation of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifying agents, disintegrants, binders, preservatives agents, buffers, lubricants and/or oils. Such excipients may optionally be included in the pharmaceutical formulation. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring and/or perfuming agents may be present in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol Sugar alcohols, inositol, sodium chloride, dry starch, corn starch, powdered sugar, etc., and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products , natural sponge, cation exchange resin, calcium carbonate, silicate, sodium carbonate, cross-linked polyvinylpyrrolidone (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked Sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate ( Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.

Exemplary surfactants and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin , gelatin, egg yolk, casein, lanolin, cholesterol, waxes and lecithin), colloidal clays such as bentonite [aluminum silicate] and [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (such as stearyl alcohol, cetyl alcohol, oleyl alcohol, triglyceride monostearate, ethylene glycol distearate, monostearyl glycerides, propylene glycol monostearate, polyvinyl alcohol), carbomers (such as carboxypolymethylene, polyacrylic acid, acrylic acid polymers and carboxyvinyl polymers), carrageenan, cellulose derivatives ( Such as sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitol fatty acid esters (such as polyoxyethylene Sorbitan monolaurate polyoxyethylene sorbitan Polyoxyethylene sorbitan monooleate Sorbitan Monopalmitate Sorbitan monostearate Sorbitan tristearate Glyceryl Monooleate, Sorbitan Monooleate Polyoxyethylene esters (eg, polyoxyethylene monostearate Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (such as ), polyoxyethylene ethers (such as polyoxyethylene lauryl ether Polyvinylpyrrolidone, Diethylene Glycol Monolaurate, Triethanolamine Oleate, Sodium Oleate, Potassium Oleate, Ethyl Oleate, Oleic Acid, Ethyl Laurate, Sodium Lauryl Sulfate, 68. Cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc., and/or combinations thereof.

Exemplary binders include, but are not limited to, starches (such as cornstarch and starch paste); gelatin; sugars (such as sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (such as acacia, sodium alginate, extracts of Irish moss, panwar gum, gum ghatta, mucilage of isapol bark, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose , hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate and larch arabinogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohols, etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, gallic acid Propyl esters, sodium ascorbate, sodium bisulfite, sodium metabisulfite and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, edetate sodium , tartaric acid and/or edetate trisodium. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetylmethylamine bromide, cetylpyridinium chloride, chlorhexidine, chlorine Butanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexetidine, amidide, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, Potassium sorbate, sodium benzoate, sodium propionate and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, alcohols, polyethylene glycols, phenols, phenolic compounds, bisphenols, chlorobutanol, parabens, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, norxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, Sodium Dialkyl Sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), Sodium Bisulfite, Sodium Metabisulfite, Potassium Sulfite, Potassium Metabisulfite, Glydant Methylparaben, Neolone ^™ , Kathon ^™ and/or

Exemplary buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, glucoheptose Calcium, calcium gluconate, d-gluconate, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxyphosphate, potassium acetate, potassium chloride , potassium gluconate, potassium mixture, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture , tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, etc., and/or combinations thereof.

Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate , sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, almond, avocado, babassu, bergamot, black tide seed, borage, juniper, chamomile, canola, coriander, carnauba, castor, cinnamon, Cocoa Butter, Coconut, Cod Liver, Coffee, Corn, Cottonseed, Emu, Eucalyptus, Evening Primrose, Fish, Flaxseed, Geraniol, Gourd, Grape Seed, Hazelnut, Hyssop, Isopropyl myristate, Jojoba, Macadamia, Brilliant Lavender, Lavender, Lemon, Litsea Cube, Macadamia, Mallow, Mango Seed, Mangosteen Seed, Mink, Nutmeg, Olive, Orange, Orange Snapper, Palm, Palm Kernel , peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, Toon, Vetiver, Walnut and Wheat Germ Oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, Isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and/or elixirs. Liquid dosage forms may contain, in addition to the active ingredient, inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate Esters, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol Fatty acid esters of diols and sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and/or perfuming agents. In certain embodiments for parenteral administration, the composition is combined with a solubilizing agent such as Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, such as sterile injectable aqueous or oily suspensions can be formulated according to known techniques using suitable dispersing agents, wetting agents and/or suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension and/or emulsification in a non-toxic parenterally acceptable diluent and/or solvent, for example a solution in 1,3-butanediol agent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. Sterile, fixed oils are generally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid are used in the preparation of injectables.

Injectable preparations can be sterilized, for example, by filtration through bacteria-retaining filters and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile sterile water or other sterile injectable media.

In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be accomplished by using liquid suspensions of poorly water soluble crystalline or amorphous materials. The rate of absorption of a drug depends upon its rate of dissolution which, in turn, depends upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable durable forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Long-lasting injectable formulations are prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are usually suppositories, which may be prepared by mixing the composition with a suitable non-irritating excipient, such as cocoa butter, polyethylene glycol or a suppository wax, which is listed in Solid at ambient temperature but liquid at body temperature, it melts and releases the active ingredient in the rectum or vaginal cavity.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient is combined with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or fillers (such as starch, lactose, sucrose, glucose, mannitol and silicic acid), binders (such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia), wetting agents (such as glycerin), disintegrants (such as agar, carbonic acid Calcium, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution blockers (such as paraffin), absorption enhancers (such as quaternary ammonium compounds), wetting agents (such as cetyl alcohol and glyceryl monohard fatty acid esters), absorbents (such as kaolin and bentonite) and lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate) and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may contain buffering agents.

Solid compositions of a similar type can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may be of a composition which release the active ingredient(s) only or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

Dosage forms for topical and/or transdermal administration of the composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. In general, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and/or any required preservatives and/or buffers as may be required. Furthermore, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and/or distributing the compound in the proper medium. Alternatively or additionally, the rate may be controlled by providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.

Suitable devices for delivering the intradermal pharmaceutical compositions described herein include short needle devices such as US Pat. Intradermal compositions may be administered by means of devices that limit the effective penetration length of a needle into the skin, such as those described in PCT Publication WO 99/34850 and functional equivalents thereof. Jet injection devices that deliver the liquid composition to the dermis via a liquid jet syringe and/or via a needle that penetrates the stratum corneum and produces a jet that reaches the dermis are suitable.射流喷射装置例如描述在美国专利5,480,381、5,599,302、5,334,144、5,993,412、5,649,912、5,569,189、5,704,911、5,383,851、5,893,397、5,466,220、5,339,163、5,312,335、5,503,627、5,064,413、5,520,639、4,596,556、4,790,824、4,941,880、4,940,460和PCT公开WO 97 /37705 and WO 97/13537. Ballistic powder/particle delivery devices that use compressed gas to accelerate the vaccine in powder form through the outer layers of the skin to the dermis are suitable. Alternatively or additionally, conventional syringes can be used for the classic Datura method of intradermal administration.

Formulations suitable for topical administration include but are not limited to liquid and/or semi-liquid formulations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments and/or pastes and/or Solutions and/or suspensions. Formulations for topical administration may, for example, contain from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the other ingredients described herein.

The pharmaceutical composition may be prepared, packaged and/or sold in a formulation suitable for pulmonary administration via the oral cavity. Such formulations may comprise dry particles comprising the active ingredient and having a diameter in the range of about 0.5 nm to about 7 nm, or about 1 nm to about 6 nm. Such compositions are conveniently in dry powder form for application using a device comprising a dry powder reservoir to which a propellant stream can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container, e.g. the device comprising a dissolving active ingredient in and/or suspended in a low-boiling propellant in a sealed container. Such a powder comprises particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nm and at least 95% of the particles have a diameter of less than 7 nm. Alternatively, at least 95% by weight of the particles have a diameter greater than 1 nm, and at least 90% by number of the particles have a diameter of less than 6 nm. Dry powder compositions may contain a solid finely divided diluent, such as sugar, and are conveniently presented in unit dosage form.

Low boiling point propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may comprise from 50% to 99.9% (w/w) of the composition and the active ingredient may comprise from 0.1% to 20% (w/w) of the composition. The propellant may further comprise other ingredients such as liquid non-ionic and/or solid anionic surfactants and/or solid diluents (which may have the same particle size as the particles comprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery may present the active ingredient in droplet form, solution and/or suspension. Such formulations may comprise aqueous and/or dilute alcoholic solutions and/or suspensions of the active ingredient, optionally sterile, prepared, packaged and/or sold and may be conveniently prepared using any nebulizing and/or nebulizing device. ground application. Such formulations may further comprise one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants and/or preservatives such as methylparaben. The droplets provided by this route of administration can have an average diameter of about 0.1 nm to about 200 nm.

The formulations described herein useful for pulmonary delivery are useful for intranasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 [mu]m to 500 [mu]m. Such formulations are administered in a snuff fashion, ie by rapid nasal inhalation from a container of powder close to the nose.

Formulations suitable for intranasal administration may, for example, contain as little as about 0.1% (w/w) and as much as 100% (w/w) active ingredient, and may contain one or more additional ingredients as described herein. In some embodiments, formulations suitable for intranasal administration comprise 0.1% to 1%, 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50% , between 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90% or 90% to 100% (w/w) active ingredient. Unless stated to the contrary, formulations suitable for intranasal administration contain 0.1-100% (w/w) active ingredient. Pharmaceutical compositions may be prepared, packaged and/or sold in formulations suitable for oral administration. Such formulations may, for example, be in the form of tablets and/or lozenges prepared using conventional methods, and may, for example, contain from 0.1% to 20% (w/w) active ingredient, the balance comprising orally dissolvable and/or dissolvable The degraded composition and optionally one or more additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise powders and/or nebulized and/or atomized solutions and/or suspensions containing the active ingredient. When dispersed, such powder, mist and/or atomized formulations may have an average particle size and/or droplet size in the range of about 0.1 nm to about 200 nm, and may further comprise one or more of the herein described any additional ingredients.

The pharmaceutical compositions may be prepared, packaged and/or sold in formulations suitable for ophthalmic administration. For example, such formulations may be in the form of eye drops comprising, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffers, salts, and/or one or more of any other additional ingredients described herein. Other ophthalmic formulations that may be used include those containing the active ingredient in microcrystalline form and/or in liposomal formulations. Ear drops and/or eye drops are contemplated to be within the scope of this invention.

General considerations in pharmaceutical formulation and/or manufacturing can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

The invention also includes pharmaceutical packs and/or kits. The provided pharmaceutical packs and/or kits can comprise the provided compositions and containers (eg, vials, ampoules, bottles, syringe and/or dispenser packs or other suitable containers). In some embodiments, provided kits may optionally further comprise a second container comprising a suitable aqueous carrier for diluting or suspending the provided compositions for preparation to be administered to an individual. In some embodiments, the contents of the provided formulation container and solvent container combine to form at least one unit dosage form.

Optionally, a single container may contain one or more compartments for containing provided compositions and/or a suitable aqueous carrier for suspension or dilution. In some embodiments, a single container can be adapted such that the container can receive physical modifications to allow for the combination of compartments and/or components of individual compartments. For example, a foil or plastic bag may comprise two or more compartments separated by a perforated seal which, upon a signal to breach the seal, may be broken to allow the combining of the contents of the two separate compartments . A pharmaceutical pack or kit may thus comprise a multi-compartment container comprising the provided composition and a suitable solvent and/or a suitable aqueous vehicle for suspension. Optionally, instructions for use are additionally provided in such kits of the invention. Such instructions will generally provide, for example, instructions for dosage and administration. In other embodiments, the instructions may further provide additional details regarding specific instructions for the particular container and/or system for administration. Still further, the instructions may provide specific instructions for use in conjunction with and/or in combination with additional therapies.

VI. Combination

In one aspect, if the biomarker diagnostics described herein predict that cortistatin or a CDK8/19 inhibitor will successfully treat the patient, it may be desirable to administer the active compound in combination with a second active agent.

In one embodiment, a sample taken from a patient is first assessed for predicted successful treatment using cortistatin or a CDK8/19 inhibitor, and then the sample is assessed in a second assay to determine whether the patient would also benefit from the effect of the second active agent. apply. In another embodiment, the results from the first biomarker assay also predict that the patient will respond to the combination therapy, as described in detail herein.

Thus, using the selection methods described herein, a treatment regimen is provided comprising combining a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog (eg, deuterated derivative) or prodrug thereof, with at least one The additional therapeutic agents are administered in combination or alternately. The combinations and/or alternations disclosed herein may be administered for beneficial, additive or synergistic effects in the treatment of abnormal cell proliferative disorders.

In specific embodiments, the treatment regimen comprises administering a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, in combination or in alternation with at least one additional kinase inhibitor. In one embodiment, said at least one additional kinase inhibitor is selected from a phosphoinositide 3-kinase (PI3K) inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, another cyclin Dependence kinase inhibitors or spleen tyrosine kinase (Syk) inhibitors or combinations thereof.

In one embodiment, a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, is combined in dosage form with a PIk3 inhibitor.

PI3k inhibitors useful in the present invention are well known. Examples of PI3 kinase inhibitors include, but are not limited to: wortmannin, demethoxyvitamin C, perifoxine, enariris, picitinib, palomid 529, ZSTK474, PWT33597, CUDC-907, and AEZS-136, duvelisib, GS-9820, GDC-0032(2-[4-[2-(2-isopropyl-5-methyl-1,2,4-triazol-3-yl)-5, 6-dihydroimidazo[1,2-d][1,4]benzoxazepan-9-yl]pyrazol-1yl]-2-methylpropionamide), MLN-1117( (2R)-1-phenoxy-2-butyl)hydro(S)-methylphosphonic acid; or methyl(oxo){[(2R)-1-phenoxy-2-butyl]oxy })), BYL-719((2S)-N1-[4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethylethyl)-4-pyridyl ]-2-thiazolyl]-1,2-pyrrolidinedicarboxamide), GSK2126458 (2,4-difluoro-N-{2-(methoxy)-5-[4-(4-pyridazinyl )-6-quinolinyl]-3-pyridyl}benzenesulfonamide), TGX-221((±)-7-methyl-2-(morpholin-4-yl)-9-(1-phenyl Aminoethyl)-pyrido[1,2-a]-pyrimidin-4-one), GSK2636771(2-methyl-1-(2-methyl-3-(trifluoromethyl)benzyl)-6 -morpholino-1H-benzo[d]imidazole-4-carboxylic acid dihydrochloride), KIN-193((R)-2-((1-(7-methyl-2-morpholino- 4-oxo-4H-pyrido[1,2a]pyrimidin-9-yl)ethyl)amino)benzoic acid), TGR-1202/RP5264, GS-9820((S)-1-(4-(( 2-(2-aminopyrimidin-5-yl)-7-methyl-4-hydroxypropan-1-one), GS-1101 (5-fluoro-3-phenyl-2-([S)]-1 -[9H-purin-6-ylamino]-propyl)-3H-quinazolin-4-one), AMG-319, GSK-2269557, SAR245409 (N-(4-(N-(3-(( 3,5-dimethoxyphenyl) amino) quinoxalin-2-yl) sulfamoyl) phenyl) -3-methoxy-4-methylbenzamide), BAY80-6946 (2- Amino-(7-methoxy-8-(3-morpholinopropoxy)-2,3-dihydroimidazo[1,2-c]quinazole), AS252424 (5-[1-[5 -(4-fluoro-2-hydroxyl-phenyl)-furan-2-yl]-methyl-(Z)-ylidene]-thiazolidine-2,4-dione), CZ24832 (5-(2-amino -8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide), Buparlisib (5-[2,6 -two( 4-morpholino)-4-pyrimidinyl]-4-(trifluoromethyl)-2-aminopyridine), GDC-0941 (2-(1H-indazol-4-yl)-6-[[4 -(methylsulfonyl)-1-piperazinyl]methyl]-4-(4-morpholinyl)thieno[3,2-d]pyrimidine), GDC-0980((S)-1-( 4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinethiophene[[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl )-2-hydroxypropan-1-one (also known as RG7422)), SF1126((8S,14S,17S)-14-(carboxymethyl)-8-(3-guanidinopropyl)-17-( Hydroxymethyl)-3,6,9,12,15-pentaoxo-1-(4-(4-oxo-8-phenyl-4H-chromen-2-yl)morpholino-4-

)-2-oxa-7,10,13,16-tetraazaoctadecane-18-ester), PF-05212384 (N-[4-[[4-(dimethylamino)-1- Piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea), LY3023414, BEZ235(2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5- c] quinolin-1-yl] phenyl} propionitrile), XL-765 (N-(3-(3-((3,5-dimethoxyphenylamino)quinoxalin-2-yl) Sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide), and GSK1059615 (5-[[4-(4-pyridyl)-6-quinolyl]methylene]- 2,4-thiazolidinedione), PX886([(3aR,6E,9S,9aR,10R,11aS)-6-[[bis(prop-2-enyl)amino]methylene]-5-hydroxy -9-(methoxymethyl)-9a,11a-dimethyl-1,4,7-trioxo-2,3,3a,9,10,11-hexahydroindeno[4,5h] isochromen-10-yl] acetate (also known as sonolisib).

BTK inhibitors useful in the present invention are well known. Examples of BTK inhibitors include: Ibrutinib (also known as PCI-32765) (Imbruvica™) (1-[(3R)-3-[4-amino-3-(4-phenoxy-phenyl)pyridine Azolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one), diphenylaminopyrimidine-based inhibitors such as AVL-101 and AVL-291/ 292(N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide) (Avila Therapeutics) (see U.S. Patent Publication No. 2011/0117073, which is incorporated herein in its entirety), Dasatinib [N-(2-chloro-6-methylphenyl)-2-(6-(4 -(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide], LFM-A13(α-cyano-β-hydroxy-β- Methyl-N-(2,5-bromophenyl)acrylamide), GDC-0834([R-N-(3-(6-(4-(1,4-Dimethyl-3-oxopiperazine- 2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetra Hydrobenzo[b]thiophene-2-carboxamide], CGI-560(4-(tert-butyl)-N-(3-(8-(phenylamino)imidazo[1,2-a]pyrazine -6-yl)phenyl)benzamide, CGI-1746 (4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6-((4-(morpholine- 4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide), CNX-774 (4-(4-((4-( (3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)-N-methylpyridinecarboxamide), CTA056(7-benzyl-1-(3-( piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one), GDC-0834((R)-N-(3-(6-((4-(1,4-Dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl -5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide) , GDC-0837 ((R)-N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl yl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide ), HM-71224, ACP-196, ONO-4059 (OnoPharmaceutica ls), PRT062607(4-((3-(2H-1,2,3-triazol-2-yl)phenyl)amino)-2-(((1R,2S)-2-aminocyclohexyl)amino )) pyrimidine-5-carboxamide hydrochloride), QL-47(1-(1-acryloylindolin-6-yl)-9-(1-methyl-1H-pyrazol-4-yl )benzo[h][1,6]naphthyridin-2(1H)-one), and RN486(6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl Base-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl )-2H-isoquinolin-1-one), and other molecules capable of inhibiting BTK activity, such as those BTK inhibitors disclosed in Akinleye et ah, Journal of Hematology & Oncology, 2013, 6:59, the entirety of which is hereby Incorporated herein by reference. In one embodiment, a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, is combined in dosage form with a BTK inhibitor.

In one embodiment, the additional cyclin-dependent kinase inhibitor is a CDK7 inhibitor, such as THZ1 (N-[3-[[5-chloro-4-(1H-indol-3-yl)pyrimidine-2 -yl]amino]phenyl]-4-[[(E)-4-(dimethylamino)but-2-enoyl]amino]benzamide). In another embodiment, the additional cyclin-dependent kinase inhibitor is a CDK9 inhibitor, such as alvocidib.

Therefore, in one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt thereof and an effective amount of a Syk inhibitor to a host in need thereof. In another embodiment, there is provided a method of treating a tumor or cancer comprising administering an effective amount of an analog of Compound A as provided herein or a pharmaceutically acceptable salt thereof in combination or alternately with an effective amount of a Syk inhibitor for hosts in need.

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of compound A or a pharmaceutically acceptable salt thereof and imatinib (Gleevec) to a host in need thereof. In another embodiment, there is provided a method of treating a tumor or cancer comprising combining an effective amount of an analog of Compound A provided herein or a pharmaceutically acceptable salt thereof with imatinib (Gleevec) or Alternately administered to hosts in need.

Syk inhibitors useful in the present invention are well known and include, for example: Cerdulatinib (4-(cyclopropylamino)-2-((4-(4-(ethylsulfonyl)piperazin-1-yl)phenyl )amino)pyrimidine-5-carboxamide), entospletinib (6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazine-8 -amine), fostamatinib ([6-({5-fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl}amino)-2,2-dimethyl- 3-oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4 base]methyl dihydrogen phosphate), fostamatinib disodium salt (6-( (5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-3-oxo-2H-pyrido[ 3,2-b][1,4]oxazin-4(3H)-yl)sodium methylphosphate), BAY61-3606 (2-(7-(3,4-dimethoxyphenyl)-imidazole [1,2-c]pyrimidin-5-ylamino)-nicotinamide HCl), RO9021 (6-[(1R,2S)-2-aminocyclohexylamino]-4-(5,6-dimethyl -pyridin-2-ylamino)-pyridazine-3-carboxylate), imatinib (Gleevec; 4-[(4-methylpiperazin-1-yl)methyl]-N-(4-methyl Base-3-{[4-(pyridin-3-yl)pyrimidin-2-yl]amino}phenyl)benzamide), staurosporine, GSK143 (2-((((3R,4R)-3 -aminotetrahydro-2H-pyran-4-yl)amino)-4-(p-tolylamino)pyrimidine-5-carboxamide), PP2(1-(tert-butyl)-3-(4-chlorobenzene base)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), PRT-060318 (2-(((1R,2S)-2-aminocyclohexyl)amino)-4-(m-toluene ylamino)pyrimidine-5-carboxamide), PRT-062607 (4-((3-(2H-1,2,3-triazol-2-yl)phenyl)amino)-2-(((1R, 2S)-2-aminocyclohexyl)amino)pyrimidine-5-carboxamide hydrochloride), R112(3,3'-((5-fluoropyrimidine-2,4-diyl)bis(azanediyl) ) diphenol), R348 (3-ethyl-4-picoline), R406 (6-((5-fluoro-2-((3,4,5-trimethoxyphenyl) amino) pyrimidine-4 -yl)amino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one), YM193306 (Singh et al.Discovery and Development ofSpleen Tyrosine Kinase(SYK)In inhibitors, J.Med.Chem.2012,55,3614-3643), 7-azaindole, picatanol, ER-27319 (see Singh et al.Discovery and Development of SpleenTyrosine Kinase (SYK) Inhibitors, J .Med.Chem.2012,55,3614-3643, the entire contents of which are incorporated herein), PRT060318 (see Singh et al.Discovery and Development of Spleen TyrosineKinase (SYK) Inhibitors, J.Med.Chem.2012,55,3614 -3643, the entire contents of which are incorporated herein), luteolin (see Singh et al.Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J.Med.Chem.2012, 55, 3614-3643, the entire contents of which are incorporated incorporated herein), apigenin (see Singhet al.Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J.Med.Chem.2012,55,3614-3643, the entire contents of which are incorporated herein), quercetin ( See (see Singh et al. Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J. Med. Chem. 2012, 55, 3614-3643, the entire contents of which are incorporated herein), fisetin (see Singh et al. Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors, J.Med.Chem.2012,55,3614-3643, the entire contents of which are incorporated herein), myricetin (see Singh et al.Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors , J.Med.Chem.2012,55,3614-3643, the entire contents of which are incorporated herein), Sambasin (see Singh et al.Discovery and Development of Spleen Tyrosine Ki nase (SYK) Inhibitors, J. Med. Chem. 2012, 55, 3614-3643, the entire contents of which are incorporated herein). In one embodiment, a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, is combined in dosage form with a Syk inhibitor.

In specific embodiments, provided methods of treatment comprise administering a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, in combination or in alternation with at least one additional chemotherapeutic agent.

In one embodiment, the at least one additional chemotherapeutic agent combined or alternated with the compound of the invention is a protein cell death-1 (PD-1 ) inhibitor. PD-1 inhibitors are known in the art and include, for example, nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech/Teva), AMP-244 (Amplimmune/GSK), BMS-936559 (BMS), and MEDI4736 ( Roche/Genentech). In one embodiment, a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, is combined with a PD-1 inhibitor in dosage form. In one embodiment, the PD-1 inhibitor is pembrolizumab.

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt thereof and an effective amount of a PD-1 inhibitor to a host in need. In another embodiment, there is provided a method of treating a tumor or cancer comprising combining or alternating an effective amount of an analog of Compound A provided herein or a pharmaceutically acceptable salt thereof with an effective amount of a PD-1 inhibitor administered to hosts in need.

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of compound A or a pharmaceutically acceptable salt thereof and pembrolizumab (Keytruda). In another embodiment, there is provided a method of treating tumor or cancer, said method comprising combining an effective amount of an analog of Compound A provided herein or a pharmaceutically acceptable salt thereof with pembrolizumab (Keytruda) or Alternate application.

In one embodiment, the at least one additional chemotherapeutic agent combined or alternated with the compound of the invention is a CTLA-4 inhibitor. CTLA-4 inhibitors are known in the art and include, for example, ipilimumab (Yervoy) marketed by Bristol-Myers Squibb and tremelimumab marketed by Pfizer.

In one embodiment, the at least one additional chemotherapeutic agent combined or alternated with the compound of the invention is a BET inhibitor. BET inhibitors are known in the art and include, for example, JQ1, I-BET151 (aka GSK1210151A), I-BET762 (aka GSK525762), OTX-015 (aka MK-8268, IUPAC 4-(4-chloro Phenyl)-N-(4-hydroxyphenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3- a][1,4]diazepine -6-acetamide), TEN-010, CPI-203, CPI-0610, RVX-208 and LY294002. In one embodiment, the BET inhibitor used in combination or alternately with the compound of the invention for the treatment of tumor or cancer is JQ1 ((S) tert-butyl 2-(4-(4-chlorophenyl)-2, 3,9-Trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine -6-yl) acetate). In another embodiment, the BET inhibitor used in combination or alternately with the compound of the present invention for the treatment of tumor or cancer is I-BET 151 (7-(3,5-dimethyl-4-isoxazolyl) -1,3-Dihydro-8-methoxy-1-[(1R)-1-(2-pyridyl)ethyl]-2H-imidazo[4,5-c]quinolin-2-one ).

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt thereof and an effective amount of a BET inhibitor to a host in need thereof. In another embodiment, there is provided a method of treating a tumor or cancer comprising combining or alternately administering an effective amount of an analog of Compound A as provided herein or a pharmaceutically acceptable salt thereof and an effective amount of a BET inhibitor for hosts in need.

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt thereof and JQ1. In another embodiment, there is provided a method of treating tumor or cancer, which comprises combining or alternately administering an effective amount of an analog of Compound A provided herein or a pharmaceutically acceptable salt thereof and JQ1.

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt thereof and I-BET 151 . In another embodiment, there is provided a method of treating a tumor or cancer comprising administering an effective amount of an analog of Compound A as provided herein or a pharmaceutically acceptable salt thereof in combination or alternately with I-BET 151 .

In one embodiment, the at least one additional chemotherapeutic agent combined or alternated with the compound of the invention is a MEK inhibitor. MEK inhibitors useful in the present invention are well known and include, for example: tametinib/GSK1120212 (N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6, 8-Dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-l(2H-yl}phenyl)acetamide), selumetinob (6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide), pimasertib/AS703026/MSC 1935369((S)-N-(2,3-dihydroxypropyl)-3-((2-fluoro-4-iodophenyl)amino)isonicotinamide), XL-518/GDC-0973(l- ({3,4-Difluoro-2-[(2-fluoro-4-iodophenyl)amino]phenyl}carbonyl)-3-[(2S)-piperidin-2-yl]azetidinine- 3-alcohol), refametinib/BAY869766/RDEA1 19(N-(3,4-difluoro-2-(2-fluoro-4-iodophenylamino)-6-methoxyphenyl)-1-(2 ,3-dihydroxypropyl)cyclopropane-1-sulfonamide), PD-0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[( 2-fluoro-4-iodophenyl) amino]-benzamide), TAK733 ((R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4- Iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione), MEK162/ARRY438162 (5-[(4-bromo-2-fluorobenzene base)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide), R05126766 (3-[[3-fluoro-2-( Methylsulfamoylamino)-4-pyridyl]methyl]-4-methyl-7-pyrimidin-2-yloxychromen-2-one), WX-554, R04987655/CH4987655 (3,4- Difluoro-2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-5-((3-oxo-1,2-oxazinane-2- base) methyl) benzamide) or AZD8330 (2-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6- Oxo-1,6-dihydropyridine-3-carboxamide). In one embodiment, a compound of the present invention or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof is combined with MEK in dosage form Inhibitor combination.

In one embodiment, the at least one additional chemotherapeutic agent combined or alternated with the compound of the invention is a Raf inhibitor. Raf inhibitors useful in the present invention are well known and include, for example: Vemurafinib (N-[3-[[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl ]carbonyl]-2,4-difluorophenyl]-1-propylsulfonamide), sorafenib tosylate (4-[4-[[4-chloro-3-(trifluoromethyl) Phenyl]carbamoylamino]phenoxy]-N-methylpyridine-2-carboxamide; 4-methylbenzenesulfonate), AZ628 (3-(2-cyanopropan-2-yl)- N-(4-methyl-3-(3-methyl-4-oxo-3,4-dihydroquinazolin-6-ylamino)phenyl)benzamide), NVP-BHG712(4- Methyl-3-(1-methyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino)-N-(3-(trifluoromethyl base) phenyl) benzamide), RAF-265 (1-methyl-5-[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]pyridin-4-yl]oxy -N-[4-(trifluoromethyl)phenyl]benzimidazol-2-amine), 2-bromoaldimine (2-bromo-6,7-dihydro-1H,5H-pyrrolo[2 ,3-c]azepine-4,8-dione), Raf kinase inhibitor IV (2-chloro-5-(2-phenyl-5-(pyridin-4-yl)-1H-imidazole-4 -yl)phenol) and sorafenib N-oxide (4-[4-[[[[4-chloro-3(trifluoromethyl)phenyl]amino]carbonyl]amino]phenoxy]-N -methyl-2-pyridinecarboxamide 1-oxide). In one embodiment, a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, is combined in dosage form with a Raf inhibitor.

In one embodiment, the at least one additional chemotherapeutic agent combined or alternated with the compound of the invention is a B-cell lymphoma 2 (Bcl-2) protein inhibitor. BCL-2 inhibitors are known in the art and include, for example: ABT-199 (4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-ene- 1-yl]methyl]piperazin-1-yl]-N-[[3-nitro-4-[[(tetrahydro-2H-pyran-4-yl)methyl]amino]phenyl]sulfonate Acyl]-2-[(1H-pyrrolo[2,3-b]pyridin-5-yl)oxy]benzamide), ABT-737 (4-[4-[[2-(4-chlorobenzene Base)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfonylbut-2-yl]amino] -3-nitrophenyl]sulfonylbenzamide), ABT-263((R)-4-(4-((4'-chloro-4,4-dimethyl-3,4,5,6 -tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio) But-2-yl)amino)-3((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide), GX15-070 (obacla methanesulfonate, (2Z)-2-[ (5Z)-5-[(3,5-Dimethyl-1H-pyrrol-2-yl)methylene]-4-methoxypyrrol-2-ylidene]indole; methanesulfonic acid))) , 2-methoxy-antimycin A3, YC137 (4-(4,9-dioxo-4,9-dihydronaphtho[2,3-d]thiazol-2-ylamino)-phenyl ester), ginkgocoumarin (pogosin), 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-benzopyran-3- Ethyl carboxylate, nilotinib-d3, TW-37(N-[4-[[2-(1,1-dimethylethyl)phenyl]sulfonyl]phenyl]-2,3, 4-trihydroxy-5-[[2-(1-methylethyl)phenyl]methyl]benzamide), apogossypolone (ApoG2) or G3139 (Oblimersen). In one embodiment, a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, is combined in dosage form with at least one BCL-2 inhibitor. In one embodiment, the at least one BCL-2 inhibitor is ABT-199 (Venetoclax).

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of compound A or a pharmaceutically acceptable salt thereof and an effective amount of a BCL-2 inhibitor to a host in need thereof. In another embodiment, there is provided a method of treating a tumor or cancer comprising combining or alternating an effective amount of an analog of Compound A or a pharmaceutically acceptable salt thereof provided herein with an effective amount of a BCL-2 inhibitor administered to hosts in need.

In one embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt thereof and ABT-199 to a host in need thereof. In another embodiment, a method for treating tumor or cancer is provided, which comprises combining or alternately administering an effective amount of Compound A or a pharmaceutically acceptable salt analog thereof provided herein and ABT-199 to patients in need Host.

In one embodiment, the treatment regimen comprises administering a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, in combination or in alternation with at least one additional chemotherapeutic agent, said additional The chemotherapeutic agent is selected from but not limited to: Imatinib mesylate (Gleevac), Dasatinib (Sprycel), Nilotinib (Tasigna), Bosutinib (Bosulif), Trastuzumab (Herceptin), Pertuzumab (PerjetaTM), lapatinib (Tykerb), gefitinib (Iressa), erlotinib (Tarceva), cetuximab (Erbitux), panitumumab (Vectibix), Vandetanib (Caprelsa), vemurafenib (Zelboraf), vorinostat (Zolinza), romidepsin (Istodax), bexarotene (Tagretin), alitretinoin (Panretin), tretinoin (Vesanoid), Carfilizomib (KyprolisTM), Pralatrexate (Folotyn), Bevacizumab (Avastin), Aflibercept (Zaltrap), Sorafenib (Nexavar), Sunitinib (Sutent), Zoparib (Votrient), regrafenib (Stivarga), and cabozolotinib (CometriqTM).

In some embodiments, the pharmaceutical combinations or compositions described herein may be administered to an individual in combination or in combination with other chemotherapeutic agents to treat a tumor or cancer. If convenient, the pharmaceutical combinations or compositions described herein may be administered concurrently with another chemotherapeutic agent to simplify the treatment regimen. In some embodiments, the pharmaceutical combination or composition and the other chemotherapeutic agent may be provided in a single formulation. In one embodiment, the pharmaceutical combinations or compositions described herein are used in combination with other agents in a therapeutic regimen. Such agents may include, but are not limited to: tamoxifen, midazolam, letrozole, bortezomib, anastrozole, goserelin, mTOR inhibitors, PI3 kinase inhibitors as described above, dual mTOR-PI3K inhibitors, MEK inhibitors as described above, RAS inhibitors, ALK inhibitors, HSP inhibitors (such as HSP70 and HSP90 inhibitors or combinations thereof), BCL-2 inhibitors as described above, induce apoptosis compounds; AKT inhibitors, including but not limited to MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4 -f][1,6]naphthyridin-3(2H)-one), GSK690693, Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Magnolol, PF-04691502 and miltefosine; PD-1 inhibitors as described above, including but not limited to Nivolumab, CT-011, MK-3475, BMS936558 and AMP-514; or FLT-3 inhibitors (including but not limited to P406, multiple Virtinib, Quizartinib (AC220), Amuvatinib (MP-470), Tandutinib (MLN518), ENMD-2076 and KW-2449), or combinations thereof.

Examples of mTOR inhibitors include, but are not limited to, rapamycin and its analogs, everolimus (Afinitor), temsirolimus, defafolimus, sirolimus, and rapamycin. Examples of RAS inhibitors include, but are not limited to, Reolysin and siG12D LODER. Reolysins of ALK inhibitors include, but are not limited to, crizotinib, AP26113, and LDK378. HSP inhibitors include, but are not limited to, geldanamycin or 17-N-allylamino-17-desmethoxygeldanamycin (17AAG) and radicicol. In a specific embodiment, a compound described herein is administered in combination with letrozole and/or tamoxifen. Other chemotherapeutic agents that may be used in combination with the compounds described herein include, but are not limited to, chemotherapeutic agents that do not require cell cycle activity for their antitumor effects.

In one embodiment, the treatment regimen comprises administering a compound of the invention, or a pharmaceutically acceptable composition, salt, isotopic analog or prodrug thereof, in combination or in alternation with at least one additional therapy, wherein the second therapy is immunotherapy therapy.

Combination drugs can be conjugated to antibodies, radiopharmaceuticals, or other targeting agents that direct the active compounds described herein to diseased or abnormally proliferating cells. In another embodiment, the pharmaceutical combination or composition is used in combination with another drug or biologic (eg, an antibody) to increase the efficacy of combined or synergistic treatment. In one embodiment, the pharmaceutical combination or composition may be used with T cell vaccination, which generally involves immunization with inactivated autoreactive T cells to eliminate cancer cell populations as described herein. In another embodiment, the pharmaceutical combination or composition is used in combination with a bispecific T cell engager (BiTE) that links two types of cells, the bispecific T cell engager (BiTE) is designed to simultaneously bind The above-mentioned endogenous T cells and cells with antibodies to specific antigens on cancer cells.

In one embodiment, the additional therapy is a monoclonal antibody (MAb). Some MAbs stimulate an immune response that destroys cancer cells. Similar to antibodies naturally produced by B cells, these MAbs "coat" the surface of cancer cells, causing their destruction by the immune system. For example, bevacizumab targets vascular endothelial growth factor (VEGF), a protein secreted by tumor cells and other cells in the tumor microenvironment that promotes the development of tumor blood vessels. When bound to bevacizumab, VEGF was unable to interact with its cell receptors, blocking the signaling that leads to the growth of new blood vessels. Similarly, cetuximab and panitumumab target epidermal growth factor receptor (EGFR), and trastuzumab targets human epidermal growth factor receptor 2 (HER-2). Monoclonal antibodies that bind to growth factor receptors on the cell surface prevent the targeted receptors from sending their normal growth-promoting signals. They can also trigger apoptosis and activate the immune system to destroy tumor cells.

Another group of cancer therapeutic MAbs are immunoconjugates. These MAbs, sometimes called immunotoxins or antibody-drug conjugates, consist of antibodies linked to cell-killing substances such as plant or bacterial toxins, chemotherapeutic drugs, or radioactive molecules. Antibodies lock onto specific antigens on the surface of cancer cells, and the cell-killing substances are taken up by the cells. FDA-approved conjugated MAbs that work in this way include ado-trastuzumab that targets the HER-2 molecule to deliver the cell-proliferation-inhibiting drug DM1 to HER-2-expressing metastatic breast cancer cells.

Immunotherapy by engineering T cells to recognize cancer cells via bispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) is an approach that has the potential to ablate both differentiated and non/slowly differentiated subpopulations of cancer cells.

By simultaneously recognizing target antigens and activating receptors on the surface of immune effector cells, bispecific antibodies offer the opportunity to redirect immune effector cells to kill cancer cells. Another approach is to generate chimeric antigen receptors by fusing extracellular antibodies to intracellular signaling domains. Chimeric antigen receptor-engineered T cells can specifically kill tumor cells in an MHC-independent manner.

In certain aspects, the additional therapy is another therapeutic agent, such as an anti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic agent, or an immunosuppressant.

Suitable chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins also known as cytotoxins or cytotoxic agents, including any agent detrimental to cell viability, and liposomes or other vesicles containing chemotherapeutic compounds.

Common anticancer drugs administered as additional agents include: vincristine (Oncovin) or liposomal vincristine (Marqibo), daunorubicin (daunomycin or glutidine) or doxorubicin (A arabinoside), L-asparaginase (Elspar) or PEG-L-asparaginase (pegasparase or pejapase) , etoposide (VP-16), teniposide (Vumon), 6-mercaptopurine (6-MP or mercaptopurine), methotrexate, cyclophosphamide (Cytoxan), prednisone, dexamethasone (Decadron), imatinib (Gleevec marketed by Novartis), dasatinib (Sprycel), nilotinib (Tasigna), bosutinib (Bosulif) and ponatinib (Iclusig ^™ ). Examples of other suitable chemotherapeutic agents include, but are not limited to: 1-dehydrotestosterone, 5-fluorouracil dacarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, doxorubicin, aldesleukin , alkylating agent, allopurinol sodium, hexamethylmelamine, amifostine, anastrozole, antramycin (AMC)), antimitotic agent, cis-dichlorodiamine platinum (II) (DDP, cisplatin ), diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, live BCG (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bolex sulfate Amycin, busulfan, leucouorin calcium, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), chlorambucil, cisplatin, carmine Drubine, colchicine, conjugated estrogen, cyclophosphamide, cyclothamide (Cyclothosphamide), cytarabine, cytarabine, cytochalasin B, cancer star, dacarbazine, actinomycin D , dactinomycin (formerly actinomycin), daunorubicin hydrochloride, daunorubicin citrate, denileukin diftitox, dexrazoxane, dibromomannitol, dihydroxyanthraxin II Ketone, docetaxel, dolasetron mesylate, doxorubicin hydrochloride, dronabinol, Escherichia coli L-asparaginase, emetine, epoetin-α, Erwinia L-asparagine Amidase, esterified estrogen, estradiol, estramustine sodium phosphate, ethidium bromide, ethinyl estradiol, etidronate sodium, etoposide citrororum factor, etoposide phosphate , filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, leucovorin, gemcitabine hydrochloride, glucocorticoids, goserelin acetate, gramicidin D, grazemi hydrochloride Agar, hydroxyurea, idarubicin hydrochloride, ifosfamide, interferon α-2b, irinotecan hydrochloride, letrozole, calcium leucovorin, leuprolide acetate, levamisole hydrochloride, lidocaine Lomustine, maytansinoid, metformin hydrochloride (mechlorethamine HCL), medroxyprogesterone acetate, megestrol acetate, melphalan hydrochloride, mercaptopurine, mesna, methylamine Pterin, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondansetron hydrochloride, paclitaxel, pamidronate disodium , pentostatin, pilocarpine hydrochloride, polymyxin (plimycin), polyphenylene 20 with carmustine implant, porphyrin sodium, procaine, procarbazine hydrochloride, propranol Er, rituximab, sargragrastim, streptozotocin, tamoxifen, paclitaxel, teniposide, tenoposide, testosterone, tetracaine, chlorambucil, thioguanine , thiotepa, topotecan hydrochloride, toremifene citrate, trastuzumab, retinoic acid, valrubicin, vinblastine sulfate, vincristine sulfate, and vinblastine tartrate Ruibin.

Suitable immunosuppressants include, but are not limited to: calcineurin inhibitors such as cyclosporine or ascomycin e.g. cyclosporine A (NEORAL), FK506 (tacrolimus), pimecrolimus, mTOR Inhibitors, such as rapamycin or its derivatives, such as sirolimus (RAPAMUNE), everolimus (Certican), temsirolimus, zotarolimus, biolimus-7, biolimus-9, rapa Pamycin (rapalog), such as defolimus, azathioprine, alemtuzumab 1H, S1P receptor modulators, such as fingolimod or its analogs, anti-IL-8 antibodies, mycophenolic acid Or its salt, such as sodium salt or its prodrug, such as mycophenolate mofetil (CELLCEPT), OKT3 (ORTHOCLONE OKT3), prednisone, ATGAM, THYMOGLOBULIN, buquina sodium, OKT4, T10B9.A-3A, 33B3 .1,15-deoxyspergualin, trepelimus, leflunomide ARAVA, CTLAI-Ig, anti-CD25, anti-IL2R, basiliximab (SIMULECT), daclizumab (ZENAPAX) , Mizoribine, Methotrexate, Dexamethasone, ISAtx-247, SDZ ASM981 (Pimecrolimus, Elidel), CTLA4Ig (Abatacept), Belatacept, LFA3Ig, Etanercept (supplied by Immunex as Enbrel marketed), adalimumab (Humira), infliximab (Remicade), anti-LFA-1 antibody, natalizumab (Antegren), ensilomab, gavilimomab, antithymocyte immunoglobulin, siplizumab , alfaccept, efalizumab, perdesian, mesalazine, oxacol, codeine phosphate, benoxylate, fenbufen, naproxen, diclofenac, etodolac, and indomel Xin, aspirin and ibuprofen.

In certain embodiments, a pharmaceutical combination or composition described herein is administered to an individual prior to, during, or after treatment with another chemotherapeutic agent, or their combination.

In some embodiments, selected drug combinations or compositions can be administered to an individual such that other chemotherapeutic agents can be administered at higher doses (increasing chemotherapeutic dose intensity) or more frequently (increasing chemotherapeutic dose density). Dose-dense chemotherapy is a chemotherapy regimen with shorter intervals between treatments than standard chemotherapy regimens. Chemotherapy dose intensity represents the unit dose of chemotherapy administered per unit of time. Dosage intensity can be increased or decreased by varying the dose administered, the interval between administrations, or both.

In one embodiment of the invention, the pharmaceutical combinations or compositions described herein may be administered in a coordinated manner with another agent, such as a non-DNA damaging, targeted antineoplastic or hematopoietic growth factor agent . It has recently been reported that inappropriate administration of hematopoietic growth factors can produce serious side effects. For example, use of growth factors of the EPO family has been associated with arterial hypertension, cerebral convulsions, hypertensive encephalopathy, thromboembolism, iron deficiency, influenza-like syndrome, and venous thrombosis. Growth factors of the G-CSF family have been implicated in splenomegaly and rupture, respiratory distress syndrome, anaphylaxis, and sickle cell complications. By combining the administration of the pharmaceutical combinations or compositions described herein with the timely administration of hematopoietic growth factors, for example, at the point in time when the affected cells are no longer in growth arrest, the health care practitioner may reduce the amount of growth factors to reduce the unwanted Minimize the side effects while achieving the desired therapeutic effect. Thus, in one embodiment, the use of the pharmaceutical combinations, compositions or methods described herein is combined with the use of hematopoietic growth factors including, but not limited to, granulocyte colony stimulating factor (G-CSF, e.g. filgrastim (filgrastin, peg-filgrastim, or legragrastim), granulocyte-macrophage colony-stimulating factor (GM-CSF, such as molastim and sargragrastim (sold by Leukine), M-CSF (macrophage colony-stimulating factor), thrombopoietin (megakaryocyte growth and development factor (MGDF), sold eg as romigrastim and eltrombopag), interleukin (IL)- 12. Interleukin-3, interleukin-11 (adipogenic inhibitory factor or interleukin oprel), SCF (stem cell factor, steel factor, kit-ligand or KL) and erythropoietin (EPO) and their derivatives ( Epoetine-alpha (sold, for example, as Darbopoetin, Epocept, Nanokine, Epofit, Epogin, Eprex and Procrit; Epoetine-beta, sold as, for example, NeoRecormon, Recormon and Micera), Epoetine-Δ (sold, for example, as Dynepo) , Epoetine-ω (sold as e.g. Epomax), Epotine-ξ (sold as e.g. Silapo and Reacrit) and e.g. Epocept, EPO Trust, Erypro Safe, Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin, Shanpoietin , Zyrop and EPIAO). In one embodiment, the pharmaceutical combination or composition is administered prior to administration of the hematopoietic growth factor. In one embodiment, the hematopoietic growth factor administration is timed such that the effect of the drug combination or composition on the HSPCs has dissipated. In one embodiment, the growth factor is administered at least 20 hours after administration of the pharmaceutical combination or composition described herein.

Multiple doses of the pharmaceutical combinations or compositions described herein can be administered to an individual, if desired. Alternatively, an individual may be administered a single dose of a pharmaceutical combination or composition described herein.

In one embodiment, the activity of the active compounds for the purposes described herein may be enhanced by conjugation with agents that target diseased or abnormally proliferating cells or otherwise enhance activity, delivery, pharmacokinetics, or other beneficial properties .

Selected compounds described herein may be administered conjugated or in combination with Fv fragments. Fv fragments are the smallest fragments produced by enzymatic cleavage of IgG and IgM class antibodies. Fv fragments have an antigen-binding site consisting of VH and VC regions, but lack CH1 and CL regions. The VH and VL chains are held together in the Fv fragment by non-covalent interactions.

In one embodiment, selected compounds described herein may be administered in combination with an antibody fragment selected from the group consisting of ScFv, domain antibody, diabody, triplet, quadruple, bi-scFv, minibody, Fab2 or Fab3 antibody fragment . In one embodiment, the antibody fragment is a ScFv. Genetic engineering methods allow the generation of single-chain variable fragments (ScFv), which are Fv-type fragments comprising VH and VL domains linked to a flexible peptide. ScFv fragments are predominantly monomeric when the linker is at least 12 residues long. Manipulation of the orientation of the V-domain and the length of the linker produces different forms of the linker of Fv molecules ranging from 3-11 residues in length, resulting in scFv molecules that cannot be folded into a functional Fv domain. These molecules can associate with a second scFv molecule to form a bivalent diabody. In one embodiment, the antibody fragment administered in combination with a selected compound described herein is a bivalent diabody. If the linker is less than three residues in length, the scFv molecules associate in triplets or quadruples. In one embodiment, the antibody fragments are triplets. In one embodiment, the antibody fragment is a quadruplet. Multivalent scFvs have greater functional binding affinity for their target antigens by binding to more than two target antigens compared to their monovalent counterparts, which reduces the off-rate of antibody fragments. In one embodiment, the antibody fragment is a minibody. Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers. In one embodiment, the antibody fragment is a bi-scFv fragment. Dual scFv fragments are bispecific. Small ScFv fragments can be generated with two different variable domains, enabling these bi-scFv molecules to bind two different epitopes simultaneously.

In one embodiment, selected compounds described herein are conjugated or administered in combination with a bispecific dimer (Fab2) or a trispecific dimer (Fab3). Genetic approaches have also been used to generate bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are capable of binding 2 (Fab2) or 3 (Fab3) different antigens at a time.

In one embodiment, selected compounds described herein are conjugated or administered in combination with rIgG antibody fragments. rIgG antibody fragment refers to reduced IgG (75,000 Daltons) or half-IgG. It is the product of selective reduction of disulfide bonds in the hinge region only. Although there are multiple disulfide bonds in IgG, those in the hinge region are the most accessible and easiest to reduce, especially with mild reducing agents such as 2-mercaptoethylamine (2-MEA). Half IgGs are usually prepared for the purpose of targeting exposed hinge region thiols that can be targeted for conjugation (antibody immobilization or enzyme labeling).

In other embodiments, selected active compounds described herein may be linked to radioisotopes to enhance efficacy using methods well known in the art. Any radioactive isotope useful to cancer cells can be incorporated into the conjugate, such as but not limited to ¹³¹ I, ¹²³ I, ¹⁹² Ir, ³² P, ⁹⁰ Sr, ¹⁹⁸ Au, ²²⁶ Ra, ⁹⁰ Y, ²⁴¹ Am, ²⁵² Cf , ⁶⁰ Co or ¹³⁷ Cs.

Notably, linker chemistry may be important for the efficacy and tolerability of drug conjugates. The thioether-linked T-DM1 has increased serum stability relative to the disulfide linker form and appears to undergo endosomal degradation leading to intracellular release of the cytotoxic agent resulting in improved efficacy and tolerability, see Barginear, M.F. and Budman, D.R., Trastuzumab-DM1: A review of the novel immune-conjugate for HER2-overexpressing breast cancer, The Open Breast Cancer Journal, 1:25-30, (2009).

Early and recent examples of antibody-drug conjugates, a discussion of the drugs that can be used in the present invention, linker chemistries, and target classes for product development can be found in the following review: Casi, G. Neri, D. , Antibody-drug conjugates: basic concepts, examples and future perspectives, J.Control Release161(2):422-428, 2012, Chari, R.V., Targeted cancer therapy: conferring specificity tocytotoxic drugs, Acc.Chem.Rev., 41(1) : 98-107, 2008, Sapra, P. and Shor, B., Monoclonal antibody-based therapies in cancer: advances and challenges, Pharmacol. Ther., 138(3): 452-69, 2013, Schliemann, C. and Neri, D., Antibody-based targeting of the tumor vasculature, Biochim. Biophys. Acta., 1776(2):175-92, 2007, Sun, Y., Yu, F., and Sun, B.W., Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue Xue Bao, 44(9):943-52, 2009, Teicher, B.A., and Chari, R.V., Antibody conjugate therapeutics: challenges and potential, Clin. Cancer Res., 17(20):6389- 97, 2011, Firer, M.A., and Gellerman, Gellerman, G.J., Targeted drug delivery forcancer therapy: the other side of antibodies, J. Hematol. Oncol., 5:70, 2012, Vlachakis, D. and Kossida, S., Antibody Drug Conjugate bioinformatics: drug delivery through the letterbox, Comput. Math. Methods Med.,2013;2013:282398,Epub 2013Jun19,Lambert,J.M.,Drug-conjugated antibodies for the treatment of cancer,Br.J.Clin.Pharmacol.,76(2):248-62,2013,Concalves,A. , Tredan, O., Villanueva, C. and Dumontet, C., Antibody-drug conjugates in oncology: from the concept totrastuzumab emtansine (T-DM1), Bull. Cancer, 99(12): 1183-1191, 2012, Newland , A.M., Brentuximab vedotin: a CD-30-directed antibody-cytotoxic drug conjugate, Pharmacotherapy, 33(1):93-104, 2013, Lopus, M., Antibody-DM1 conjugates as cancer therapeutics, Cancer Lett., 307(2) :113-118, 2011, Chu, Y.W. and Poison, A., Antibody-drug conjugates for the treatment of B-cell non-Hodgkin's lymphoma andleukemia, Future Oncol., 9(3):355-368, 2013, Bertholjotti, I., Antibody-drugconjugate a new age for personalized cancer treatment, Chimia, 65(9):746-748, 2011, Vincent, K.J., and Zurini, M., Current strategies in antibody engineering: Fcengineering and pH–dependent antigen binding , bispecific antibodies and antibody drug conjugates, Biotechnol.J., 7(12):1444-1450, 2012, Haeuw, J.F., Caussanel, V., and Beck, A., Imm unoconjugates, drug-armed antibodies to fight against cancer, Med.Sci., 25(12):1046-1052, 2009; and Govindan, S.V., and Goldenberg, D.M., Designing immunoconjugates for cancer therapy, Expert Opin. Biol. Ther., 12(7):873-890, 2012.

In one embodiment, the pharmaceutical compositions or combinations described herein are useful in the treatment of any of the disorders described herein.

In one aspect, a compound of the invention is administered in combination or composition with an effective amount of a nucleoside or nucleoside analog. Non-limiting examples of nucleosides include: azacitidine, decitabine, didanosine, vidarabine, BCX4430, cytarabine, emtricitabine, lamivudine, zalcitabine , abacavir, acyclovir, entecavir, stavudine, telbivudine, zidovudine, iodovoxatidine, trifluorouracil, aprecitabine, elvucitabine, amdosovir and racivir. In one embodiment, a compound of the present invention is used in combination or composition with an effective amount of a nucleoside or nucleoside analog for the treatment of a viral infection. In an alternative embodiment, a compound of the invention is used in combination or composition with an effective amount of a nucleoside or nucleoside analog for the treatment of tumors or cancer. In one embodiment, the nucleoside analog is azacitidine and the disorder is a tumor or cancer.

In one embodiment, a method of treating a tumor or cancer in an individual is provided, comprising administering Compound A or a pharmaceutically acceptable salt thereof in combination or alternately with an effective amount of a nucleoside analog to a host in need thereof. In another embodiment, there is provided a method of treating a tumor or cancer in an individual comprising combining or alternately administering an analog of Compound A or a pharmaceutically acceptable salt thereof provided herein and an effective amount of a nucleoside analog to A host in need.

In one embodiment, there is provided a method of treating a tumor or cancer in an individual comprising administering Compound A, or a pharmaceutically acceptable salt thereof, in combination or alternately with azacitidine to a host in need thereof. In another embodiment, there is provided a method of treating a tumor or cancer in an individual comprising administering an analog of Compound A or a pharmaceutically acceptable salt thereof provided herein in combination or alternately with azacitidine to an individual in need thereof. Host.

VII. Embodiment

Example 1: Determination of genes associated with RUNX1-RUNX1T1 in Kasumi-1 cells

Kasumi-1AML cells contain the RUNX1-RUNX1T1 fusion protein. A genome was obtained that determined genes in Kasumi-1 cells whose expression increased upon RUNX1-RUNX1T1 knockdown (Ben-Ami, O. et al. Cell Reports 4, 1131-1143 (2013)). Using genome enrichment analysis (Subramanian, A. et. al. Proc. Natl Acad. Sci. USA102, 15545–15550 (2005)), this genome was combined with an extensive molecular marker database (C2) with 25 nM cortistatin A comparison of genes differentially expressed in MOLM-14 cells after 3 hours of treatment (Fig. 2).

Example 2: Determination of gene expression levels

Leukemic cells were plated in triplicate (12 wells) at 500,000-800,000 cells per ml and incubated in vehicle (0.1% DMSO) or CA (for K562, MOLM-14 and MV4; 11, 25 nM, 3 hours; for MOLM- 14 for 10 nM for 24 hours; for SET-2 for 25 nM for 4 hours, incubated in the presence of n=3 for each cell line). Cells were then washed twice with cold PBS and snap frozen. RNA was isolated (RNeasy Plus Microkit, Qiagen or TRIzol, Life Technologies), processed, and for K562, MOLM-14 and MV4;11, hybridized to human U133Plus 2.0 microarrays (Affymetrix). Microarrays were processed using Bioconductorpackages affyQCReport for quality control and affy for background correction, pooling and normalization using RMA. Probesets present in at least 1 sample (based on affymas5call) and whose interquartile range > log ₂ (1.2) were retained for further analysis. Differential expression analysis of CA-treated and DMSO control samples was performed using the limma Bioconductor software package (P<0.05 with Benjamini-Hochberg correction). SET-2 and HCT116 gene expression was measured by RNA-seq. SET-2 RNA-seq libraries were prepared and processed using the Ion Torrent workflow. Reads were aligned twice, first with rnaStar (v.2.3.0e) and then with BWA (v.0.7.5a) for the remaining unmapped reads, both using default parameters. Mapped reads were pooled and counted using HTSeq (v.0.5.3p3) with -s yes-m intersection-strict. DE analysis (FDR<0.05, double change) and normalization were performed using the Bioconductor software package DESeq. HCT116 cells were grown to approximately 80% confluency and treated with 100 nM CA or DMSO for 3 hours (n=3). Cells were then washed twice with cold PBS and scraped into TRIzol reagent (Life Technologies). After RNA was collected, it was further purified using RNeasy mini kit (Qiagen) and on-column DNase I digestion. Libraries for Illumina sequencing were generated with the Illumina TruSEQ Stranded mRNA Prep Kit. Samples were run on a single lane of an Illumina HiSEQ 2000 sequencer with a single read flow cell using 1 50-bp read and 6 cycle index reads. Reads were mapped to the hg19 reference genome using Tophat2v.2.0.6 with custom settings including the setting -library-type fr-firstrand to properly account for the retention nature of the protocol. Read counts for annotated genes were obtained using HTSeq v.0.6.1, and differentially expressed genes were called by DESeq v.1.10.1 with padj values less than 0.01. Counts were normalized for use in GSEA using the limma voom function. Expression data for I-BET151 comparisons were downloaded from ArrayExpress (https://www.ebi.ac.uk/arrayexpress, accession number E-MTAB-774) and processed files were used as received. Submit the gene list to the DAVID web server (http://david.abcc.ncifcrf.gov) for functional annotation. GSEA version 2.09 is performed using the natural-valued signal-to-noise ratio as the measure. Signatures included curated gene sets (C2, v.3) downloaded from Broad's MSigDB as well as signatures curated from internal and published datasets. alpha

The results of the gene expression screen are summarized in Table 2 below.

*MOLM-14 cells, treated with 3hCA; **SET-2 cells, treated with 4hCA

Table 2: Cortistatin A upregulates RUNX1 target genes. GSEA indicates upregulation of the RUNX1 target gene signature following 3 h 25 nM cortistatin A treatment in MOLM-14 cells or 4 h 25 nM cortistatin A treatment in SET-2 cells.

Using genomic enrichment analysis, we found that treatment of MOLM-14 cells or SET-2 cells increased the expression of these RUNX1 target genes. Curated genomes are analyzed together with more than 4,000 tags including the Extensive Molecular Tag Database (C2).

Example 3: Method for Determining Differentiation of Cells of Study

For the SET-2 differentiation assay, cells were plated at 150,000 cells/ml in triplicate (6 wells) for 3 days with 50 nM CA, 50 ng ml ⁻¹ PMA (positive control) or vehicle. Cell pellets were collected at 4°C, washed three times with cold PBS, stained with anti-CD61-PE (ab91128) or anti-CD41-PerCP (ab134373) and analyzed by flow cytometry. For each experiment, n=3 biological replicates with two independent experiments, one shown (Figure 3).

Example 4: Cell Proliferation Assay

All suspension cells were plated in triplicate (96 wells) at 5000-30000 cells per well for testing (n=3). Viable cell numbers were estimated after 3, 7, and 10 days by counting viable cells from one medium well, generating a cell dilution series, transferring 20 ml per well in duplicate to a 384-well plate, and using CellTiter-Glo (Promega) Response (SPECTRAmaxM3, MoleculaRDevices) for linear regression. Cells from all wells were also diluted fourfold in culture medium and transferred in duplicate for CellTiter-Glo measurements. On days 3 and 7, equal volumes of all wells were split with fresh medium and compound such that the resulting cell density in the medium matched the initial seeding density. For days 7 and 10, estimated cell numbers represent division-adjusted theoretical cell numbers. HCT116 (96 wells) were plated in triplicate at 250 cells per well. Cells were incubated in the presence of vehicle, 1 [mu]M paclitaxel or compound. On day 7, CellTiter-Blue (Promega) responses were measured and values were normalized to vehicle (100% growth) and paclitaxel (0% growth). For growth assays with inhibitors, n=3 per concentration, with two independent experiments.

The results are summarized in Table 3 below, a graph showing proliferation over time (Figure 4).

Table 3: Sensitivity of selected cortistatin A cell lines.

Table 3 shows that a number of blood cancer cell lines were inhibited in growth by the CDK8/19 inhibitor cortistatin A. Two of the highly sensitive cell lines had mutations in RUNX1 itself (SKNO-1 and REH) and possibly others with reduced levels of RUNX1 (RS4; 11, MV4; 11 and MOLM-14); based on finding MLL fusions Decrease the protein level of RUNX1 (Zhao, X. et al. Downregulation of RUNX1/CBFβ by MLL fusion proteins enhances hematopoieticstem cell self-renewal. Blood 123, 1729–1738 (2014))). Other cell lines are expected to be sensitive to cortistatin A because cortistatin A increases the RUNX1 transcriptional program. These include the megakaryocyte lines MOLM-16, SET-2, MEG-01 and CMK-86, as RUNX1 is a megakaryocyte (de Bruijn, M.F. & Speck, N.A. Core-binding factors inhematopoiesis and immune function. Oncogene 23, 4238–4248( 2004)) and ALL-SIL differentiation of ALL cell lines with overexpression of TLX1. TLX1 overexpression was shown to control the RUNX1 transcriptional program (Gatta, Della, G. et al. Reverse engineering of TLX oncogenic transcriptional network identifies. Nat. Med. 18, 436–440 (2012)).

Cortistatin potently inhibits the proliferation of many AML cell lines with a ₅₀ % maximal growth inhibitory concentration (GI50) of less than 1OnM. Cell line sensitivity is consistent with RUNX1 transcriptional program dependence. Sensitive cell lines include those containing fusions that directly inhibit RUNX1 or its target genes (SKNO-1, ME-1, MOLM-14) as well as megakaryocytic leukemia cell lines with truncated GATA-1 protein GATA-1s (CMK -86 and MEG-01). Unlike megakaryopoiesis, RUNX1 expression rapidly declines during erythroid terminal differentiation, consistent with the insensitivity of erythroleukemic cell lines to CA. For example, it was determined that corticosterone increases the RUNX1 transcriptional program in the AML cell lines SET-2, MOLM-14 and MV4;11. Cortistatin upregulates RUNX1 target genes, including CEBPA, IRF8, and NFE2, and was determined by Genome Enrichment Analysis (GSEA) to (i) cortistatin upregulates SET-2, MOLM-14, and MV4; genes in 11 cell lines, which Inhibited by expression of RUNX1-RUNX1T1 in hematopoietic stem cells; (ii) sebastatin upregulates MOLM-14 and MV4;11 genes in cells whose expression was reduced in the Kasumi-1 AML cell line upon siRNA-mediated knockdown of RUNX1 and (iii) sebastatin upregulates genes in MOLM-14 cells whose expression is increased in Kasumi-1 cells upon siRNA-mediated knockdown of RUNX1-RUNX1T1. RUNX1 is recruited to sites upregulated by cortistatin treatment.

Example 5: SET-2/UKE-1 Synergy Test

SET-2 and UKE-1 cells were co-treated with a constant ratio of ruxolitinib to CA, 1:1 or 10:1, in a 96-well growth assay format with a range of 2-fold dose dilutions of the compound. SET-2 and UKE-1 cells were also treated with ruxolitinib alone or CA alone in a 2-fold dilution series. The Chou-Talalay combination index value at 50% growth inhibition (Fa=0.5) was determined using CalcuSyn software (see Chou, T.C. Cancer Res. 2010 Jan 15;70(2):440-6) (Figure 5).

Example 6: In vivo xenograft studies

The MV4;11 xenograft model was performed as previously described (Etchin, J. et al. Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells. Leukemia 27, 66-74 (2013)). Two million MV4;11-mCLP cells were injected into the tail vein of 7-week-old female non-obese diabetic severe combined immunodeficiency (NOD-SCID) Il2rg ^-/- (NSG) mice (Jackson Laboratories) and analyzed using the IVIS spectroscopy system ( Caliper Life Sciences) assessed tumor burden by bioluminescence imaging (BLI). Seven days post-injection, the establishment of leukemia was documented by BLI, and mice were grouped to achieve a similar mean BLI and treated with vehicle (20% hydroxypropyl-β-cyclodextrin) or CA once daily ip for 15 days. After 30 days, blood cell counts (Hemavet 950F, Drew Scientific) were obtained, and spleen, femur, and peripheral blood cells were collected from three mice in each group and analyzed by flow cytometry (LSRFortessa, BD Biosciences), with the highest, lowest, and median BLI value. After weighing the spleen (Figure 6) and opening the body cavity and exposing the internal organs, the mouse and a portion of the spleen were preserved in fixative. Samples from all organs were then dissected and placed into nine boxes per mouse. Tissues were embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin. Survival was measured from initiation of treatment until the moribund state. Statistical analysis was performed using GraphPad Prism 6.0. For P-value determination, use two-way or one-way ANOVA together with Dunnett's multiple comparison test and P-value adjustment.

Example 7: Analysis of native and recombinant kinases

Native kinase assays were performed with MOLM-14 cell lysates according to the KiNativ method from ActivX Biosciences. For each quantified peptide, the change in the mass spectral signal of the treated samples relative to the signal of the control samples was expressed as percent inhibition. Results correspond to one experiment in duplicate for each cortistatin A (CA) concentration. The percent change in mass spectrometric signal reported was statistically significant (Student's t-test score <0.04) (Figure 7A and Figure 7B).

Recombinant holokinome selectivity analysis. A radioactive protein kinase assay (PanQinase activity assay; performed by ProQinase GmbH) was used as described (Hutterer, C. et al. Antimicrob. Agents Chemother. 59, 2062-2071 (2015)).

Example 8. In vitro radioactive protein kinase assay

A radioactive protein kinase assay (PanQinase activity assay; performed by ProQinase GmbH) was used as described by Hutterer, C. et al. Antimicrob. Agents Chemother. 59, 2062-2071 (2015). The IC50 determination of CDK8-CCNC (8.3nM, 1.0μM ATP and 1.0μg/50ml substrate RBER-IRStide) was performed as a repeated measurement, and the _IC50 was calculated using Prism 5.04 with a sigmoidal response, with the top fixed at 100 _% , and the bottom at 0% of the least squares fit (Figure 8).

Example 9: Screening for drug resistance alleles

5'-Flag-tagged CDK8 and CDK19 were cloned from pBabe.puro.CDK8.flag (Addgene19758) and F-CDK8L (Addgene24762) into pLVX-EF1α-IRES-mCherry and pLVX-EF1α-IRES-ZsGreen (Clontech) and transformed into coli (One Shot Stb13, Invitrogen). Point mutations were introduced by whole-plasmid PCR (QuikChange II XLSite-Directed Mutagenesis Kit, Agilent). The pLVX lentiviral vector was co-transfected with psPASx and pMD2.G (Addgene) in 293T cells. After 48 hours, viral supernatants were collected and passed through a 0.45 μm filter (Millipore). For transduction, 24-well plates were coated with 500 μl 20 μg/ml RetroNectin (Clontech) overnight at 4° C., blocked with 2% BSA for 30 minutes, washed with PBS, and 300-500 μl viral supernatant was added. Plates were centrifuged (2,000 g, 1.5 h) and placed in an incubator. After 2 hours, the viral supernatant was removed and 200,000 cells per ml were added in 500 μl per well. After 1-3 days, cells were expanded and isolated by FACS.

Drug-resistant alleles demonstrate that CDK8/19 kinase activity is required for AML cell growth. This suggests that the CDK8/19 inhibitor cortistatin A inhibits the proliferation of MOLM-14 cells by inhibiting CDK8/19. Mutation of tryptophan 105 (W105) in CDK8 and CDK19 confers cortistatin A resistance to CDK8 and CDK19. Thus, MOLM-14 cells were able to proliferate in the presence of cortistatin A upon expression of CDK8W105M or CDK19W105M.

Example 10: CDK8/19 inhibition prevents leukemia cell growth in vivo.

The MV4;11 xenograft model was performed as previously described (Etchin, J. et al. Antileukemic activity of nuclear export inhibitors that spare normal hematopoietic cells. Leukemia 27, 66-74 (2013)). Two million MV4;11-mCLP cells were injected into the tail vein of 7-week-old female non-obese diabetic severe combined immunodeficiency (NOD-SCID) Il2rg ^-/- (NSG) mice (Jackson Laboratories) and analyzed using the IVIS spectroscopy system ( Caliper Life Sciences) assessed tumor burden by bioluminescence imaging (BLI). Seven days post-injection, the establishment of leukemia was documented by BLI, and mice were grouped to achieve a similar mean BLI and treated with vehicle (20% hydroxypropyl-β-cyclodextrin) or CA once daily ip for 15 days. After 30 days, blood cell counts (Hemavet 950F, Drew Scientific) were obtained, and spleen, femur, and peripheral blood cells were collected from three mice in each group and analyzed by flow cytometry (LSRFortessa, BD Biosciences), with the highest, lowest, and median BLI value. After weighing the spleen (Figure 6) and opening the body cavity and exposing the internal organs, the mouse and a portion of the spleen were preserved in fixative. Samples from all organs were then dissected and placed into nine boxes per mouse. Tissues were embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin. Survival was measured from initiation of treatment until the moribund state. Statistical analysis was performed using GraphPad Prism 6.0. For P-value determination, two-way or one-way ANOVA was used with Dunnett's multiple comparisons test and P-value adjustment.

Analysis of MV4;11 AML mice at day 30 showed that treatment with the CDK8/19 inhibitor cortistatin A resulted in fewer leukemic cells in the lungs, as measured by hematoxylin and eosin staining (Figure 10).

Example 11: CDK8/19 inhibition in AML/megakaryocyte lines increases the expression of RUNX1 target genes and recruits RUNX1 to specific genomic loci.

Determined that CA increases the RUNX1 transcriptional program in the CA-sensitive cell lines SET-2, MOLM-14, and MV4;11. CA upregulates RUNX1 target genes, including CEBPA, IRF8, and NFE2, and was determined by Genome Enrichment Analysis (GSEA):

1. CA upregulates SET-2, MOLM-14, and MV4;11 genes in the cell line, which are repressed by the expression of RUNX1-RUNX1T1 in hematopoietic stem cells (HSCs) (Figure 2). The RUNX1-RUNX1T1 fusion largely functions to repress the transcription of RUNX1 target genes.

2. CA upregulates genes in MOLM-14 and MV4;11 cells, whose expression is reduced in Kasumi-1 AML cell line upon siRNA-mediated knockdown of RUNX1.

3. CA upregulates genes in MOLM-14 cells, whose expression increases in Kasumi-1 cells upon siRNA-mediated RUNX1-RUNX1T1 knockdown.

Mechanistically, RUNX1 was observed to be recruited to loci upregulated by CA treatment (Fig. 11), suggesting that CDK8/19 kinase activity restricts RUNX1 from target loci, preventing increased expression of RUNX1 target genes.

Example 12: RUNX1 alterations and associated mutations as predictive biomarkers for CDK8/19 inhibitor treatment.

The antiproliferative activity and effect on differentiation of cortistatin A (CA) or analogs thereof can be measured in primary patient samples. Patient samples (20-40) that have been characterized as containing mutations or translocations in RUNX1, including RUNX1-RUNX1T1 and monoallelic, loss-of-function RUNX1 point mutants, were obtained. 10-30 additional patient samples were also obtained. It has mutations in transcriptional regulators that together with RUNX1 regulate RUNX1 target genes, including mutations in GATA1 exon 2, CBFb-MYH11 translocation, FUS-ERG translocation, and loss-of-function CEBPA indel mutants. To assess sensitivity to CA or its analogs, 3-day liquid culture and clonogenic assays were performed on unfractionated and CD34+ leukemic cells from patients. For clonogenic assays, 1,000-2,000 cells per well were plated in duplicate in media supplemented with human cytokines (rhSCF, rhG-CSF, rhGM-CSF) in the presence of vehicle, CA or CA analogs (100 nM, 20 nM and 4 nM). , rhIL-3, rhIL-6, rhEpo) in Methocult (Stem Cell Tech, H4435). After 14 days of incubation at 37°C, the total colonies on each plate were counted. In parallel, CD34+ cells were treated with vehicle, CA or CA analogs in the presence of cytokines for 5 days and then labeled with myeloid markers for differentiation analysis by flow cytometry. Considering that each patient sample represents a limited qualifier resource, in initial testing cells that had undergone CDK8/19 inhibitor and vehicle treatment were collected for subsequent genomic analysis, such as gene expression studies. These samples will help validate hits from CRISPR-Cas9 screens.

Using the methods described above, a large number of primary AML patient samples representing the most common genetic alterations in AML7, including mutations in DNMT3A, NPM1, WT1, components of the cohesion complex, and FLT3, were evaluated.

Example 13: CRISPR-Cas9. Cas9 validation and initial screening in MOLM-14 cells

Human Streptococcus pyogenes (S.pyogenes) Cas9 was expressed in the CA-sensitive AML cell line MOLM-14 and its ability to knock out two genes was confirmed: ZsGREEN (a lentiviral integrated gene encoding green fluorescent protein) and BCL2L11 (gene encoding the pro-apoptotic endogenous protein Bim) (Figure 12 and Figure 13).

A CRISPR-Cas9 modifier screen in MOLM-14 cells was performed to identify knockout genes conferring resistance to CDK8/19 inhibition. MOLM-14 cells were transduced in triplicate with a broad lentiviral library encoding 80,000 sgRNAs targeting 18,000 genes in the human genome (4 sgRNAs/gene plus a control sgRNA) and linked to puromycin resistance Marker co-expression. Cells expressing both Cas9 and sgRNA on blasticidin and puromycin for seven days were selected before starting the selection. The workflow is depicted in Figure 14. Changes in sgRNA distribution from day 0 to day 14 were compared between vehicle and CA-treated groups. Guides were enriched in the CA group but not the vehicle group, representing positive regulators of CA sensitivity. Genes were ranked according to the RIGER score, considering not only the fold-change difference between the two treatment groups, but also the reproducibility between replicates and the similarity of action between redundant sgRNAs. Top hits were verified by knocking out the gene using CRISPR-Cas9, validating the knockout using western blotting or qPCR, and measuring resistance to CA, respectively.

Example 14: Determining the sensitivity of primary pediatric patient AMKL cells to CDK8/19 inhibition in vitro and in a mouse xenograft model

The antiproliferative activity and effect on differentiation of cortistatin A (CA) or analogs thereof in primary patient AMKL samples can be determined. Collect 10 to 30 patient samples first, including pediatric DS-AMKL and pediatric non-DS-AMKL who have been characterized for genetic lesions such as GATA1 status and the presence of common translocations in non-DS-AMKL, common translocations such as MLL rearrangements, RBM15 -MKL1, CBFA2T3-GLIS2 and NUP98-KDM5A. Samples expanded first in sublethally irradiated NOD.Cg-Prkdc ^Il2rgl /SzJ(NSG) mice were then prioritized so that subsequent testing in mouse xenograft models could be performed if sensitive in vitro . For patient samples that had not been expanded in vivo, transplantation and expansion in NSG mice was first attempted.

For in vitro testing, AMKL cells were cultured with CA, its analogs or vehicle for 3-5 days. Cell numbers were then measured over time, and flow cytometry was used to characterize cellular effects of (1) changes in megakaryocyte-specific markers CD41 and CD42, (2) ploidy changes, and (3) induction of apoptosis. These in vitro experiments were previously described for the Aurora kinase A inhibitor MLN8237. Up to 5 patient samples that were sensitive to CDK8/19 inhibition in vitro and had been transplanted and expanded in NSG mice were subsequently tested in an in vivo xenograft model following the procedure used for testing MLN8237. Specifically, patient leukemic blasts from primary, secondary or tertiary recipient mice were injected intrafemorally into sublethally irradiated NSG mice (7 per treatment group). )middle. After an engraftment period of approximately 10 days, mice were treated with CA, its analogs, or vehicle for 15 days, followed by sampling of bone marrow (days 27 and 70) and peripheral blood (day 55). Monitor disease burden and differentiation. Disease burden was measured by flow cytometry at all time points by human CD45 expression to determine the presence of human cells, and differentiation was measured by human CD42 expression 3 days after treatment. In addition to sampling, mice were monitored for hind leg paralysis and survival.

Example 15: Determining whether CDK8/19 inhibition restores the RUNX1 transcriptional program in AMKL.

For 3-5 CA-sensitive leukemic blasts from patients described in Example 14, gene expression and RUNX1 occupancy after treatment with CA, its analogs or vehicle were measured. RUNX1 occupancy was measured by chromatin immunoprecipitation followed by sequencing (ChIP-seq). These experiments paralleled those performed with the SET-2 megakaryocyte line and were able to demonstrate that (1) CA or its analogues stimulate the RUNX1 transcriptional program in these patient leukemia cells, and (2) by rescuing hollow blocks in RUNX1 recruitment to specific genomic loci that are also transcriptionally upregulated. In addition, gene expression was measured in up to three CA-resistant AMKL patient samples to determine whether modulation of the RUNX1 transcriptional program was selectively observed in sensitive cells. Comparing the underlying gene expression patterns of sensitive and insensitive AMKL patients allows determining whether certain gene expression programs are associated with sensitivity.

Example 16: Genome-wide CRISPR-Cas9 modifier screen to identify predictive biomarkers for CDK8/19 inhibitor therapy

A genome-wide CRISPR-Cas9 modifier screen was performed to identify genetic alterations in AML that might predict susceptibility. Screening occurred in cortistatin A (CA) sensitive AML cell lines (>50% growth inhibition at 100 nM CA) and CA insensitive AML cell lines (<50% growth inhibition at 100 nM CA). For CA-sensitive cell lines, genes were identified that, when knocked out, confer CA resistance, and likewise in CA-insensitive cell lines, genes were identified that, when knocked out, confer CA sensitivity. By testing multiple cell lines, cell line-specific genetic alterations can be ruled out, and by testing CA-sensitive and CA-insensitive cell lines, patterns of genetic alterations that predict sensitivity to CA or CA analogs can be identified. The results of this screen can be used to assess gene expression levels in AML patient samples for which sensitivity to CDK8/19 inhibitors has been assessed.

Example 17: Screening of 62 Cells for Cortistatin A Sensitivity Using Various Biomarkers

For 62 cell lines, 96-well suspension cell culture plates were prepared. 100 [mu]L of soft agar bottom layer (0.6% final concentration in complete medium) was poured and solidified. Then 50 μL of the top layer of soft agar (0.4% final concentration) containing the corresponding cells and cell numbers was added on top, allowed to solidify and incubated at 37°C, 10% CO ₂ . After the soft agar had solidified, the test substances were added to the inner wells of the plates at the indicated final concentrations. Subsequently, assays were incubated in a cell culture incubator for 8-14 days. Finally, the assays were developed using Alamar Blue and the fluorescence intensity (excitation: 560nm; emission: 590nm) was measured after incubation at 37°C for 1-5 hours. As a low control, cells were treated with 1E-05M staurosporine (6-fold value). As a high control, cells were treated with 0.1% DMSO (solvent control, 6-fold value).

Raw data were converted to soft agar growth percentages relative to the high control (solvent 0.1% DMSO) and low control (1E-05M staurosporine), which were set at 100% and 0%, respectively. The results are presented in Figures 15A, 15B and 15C and Table 4 below.

Table 4: Growth % of the 15 most inhibited cell lines tested, and corresponding biomarkers present.

Example 18: Cortistatin for the Treatment of Myelodysplastic Syndrome (MDS)

Cortistatin A significantly increased the expression of a number of RUNX1-target genes, including CEBPA, IRF8 and NFE2, in AML cell lines, including the MOLM-14 cell line derived from MDS patients (Figure 2, Example 1). RUNX1 is known to be mutated in 10-20% of MDS patients and is the most frequently mutated gene in MDS. Therefore, cortistatin and its analogs can effectively treat MDS by increasing the expression of the RUNX1 gene. Further confirmation is provided by the following experimental results: 1) CA treatment upregulates CA-induced RUNX1 recruitment to loci, suggesting that CDK8/19 kinase activity blocks RUNX1 accumulation at target loci (Example 11), and 2) CA treatment The antiproliferative activity of RUNX1 was positively correlated with cell lines with impaired expression of RUNX1 target genes, including those with RUNX1 mutations.

Example 19: Megakaryotic cell lines are highly sensitive to the CDK8/19 inhibitor cortistatin A (CA), and CA antiproliferative activity is consistent with stimulation of the RUNX1 transcriptional program.

Cortistatin A (CA) was found to potently inhibit the proliferation of the five megakaryocyte lines tested with a ₅₀ % maximal growth inhibitory concentration (GI50) of less than 10 nM (Table 5). Among the cell lines tested was CMK-86, which contains a truncated protein of GATA1s and was derived from a pediatric DS-AMKL patient.

Table 5. Sensitivity of leukemia cell lines to CA consistent with RUNX1 dependence, _GI50 exhibited after 10 days of treatment. Note the marked sensitivity of many megakaryocyte lines.

The antiproliferative activity of CA in leukemia cell lines matches its expected dependence on dysregulation of the RUNX1 transcriptional program. In addition to megakaryocyte lines, CA potently inhibited the proliferation of cell lines containing chimeric proteins (fusions) that directly inhibit the transcription of RUNX1 or its target genes (Table 5), including those containing the RUNX1-RUNX1T1 fusion Cell lines and cell lines containing MLL fusions. Furthermore, erythroid and leukemic cell lines were insensitive to CA, consistent with decreased RUNX1 expression in erythroid differentiation (lack of RUNX1 dependence in terminal erythroid differentiation). The strong lineage-dependent sensitivity of the cell lines to CA is especially evident in the results that the sensitivity of megakaryocyte and erythroleukemic cell lines harboring the same mutation (BCR-Abl or JAK2V617F) differed by more than 100-fold (compare K562 and HEL with MEG-01 and SET-2).

This specification has been described with reference to the embodiments of the present invention. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the present invention as set forth in the claims. Accordingly, the specification is to be regarded as illustrative rather than restrictive, and all such modifications are intended to be included within the scope of present invention.

Claims (79)

1. for targetting selection and treatment tumour or the method for cancer patient, it includes (i) and determines whether the patient has RUNX1 approach is damaged；If it has, (ii) optionally with pharmaceutically acceptable composition apply effective dose cortex chalone or Its pharmaceutically acceptable salt or oxide.

2. the method for treating the impaired tumour of RUNX1 in patient or cancer, it includes what is generally transcribed to produce by RUNX1 The mode and dosage of the abundant up-regulation of albumen apply the cortex chalone of effective dose, so that cell more normal, virulence are weaker, more ripe Or the mode of prevention growth or apoptosis causes the differentiation of the tumour or cancer.

3. method according to claim 1 or 2, also including the use of for determining whether the patient will be successfully responding to skin The kit of matter chalone treatment, exists wherein the kit includes with the polynucleotides of biomarker or biomarker combinations The probe or the antibody of combination biomarker protein annealed under stringent condition.

4. for predicting the patient with tumour or cancer to the method for the response treated with cortex chalone, it includes：

I. tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. determine that the expression of assessment or expression quantity are whether outside the scope of corresponding normal cell in step (ii), example Such as, higher or lower than the scope found in corresponding normal cell, or higher or lower than with the increase of the clinical Benefit of patient Or reduce a certain amount of of correlation；With

Iv. optionally optionally pharmaceutically may be used with the cortex chalone of effective dose or its pharmaceutically acceptable salt or oxide with it Patient described in the composition treatment of receiving.

5. the method for the patient with tumour or cancer for being suitable for being treated with cortex chalone for selection, methods described include：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, with described in determination Whether patient has response to the treatment of cortex chalone, and the control sample has response including representative number to cortex chalone Patient or predictive animal model and representative number to cortex chalone without or have the patient of poor response；With

Iv. if it is determined that the patient has response to the treatment, then optionally being applied with its pharmaceutically acceptable composition has The cortex chalone or its pharmaceutically acceptable salt or oxide of effect amount.

6. according to the method any one of claim 1-5, in addition to for assessing impaired selected of diagnosis RUNX1 approach The kit of the expression of gene, wherein the kit is mutual comprising the RNA for being used to expand and the gene specific encodes The DNA of benefit primer and optional heat-staple archaeal dna polymerase.

7. according to the method for claim 6, wherein every kind of primer under standard stringent condition with the selected genes The RNA of coding or its complementary sequence hybridization.

8. according to the method any one of claim 1-7, wherein selected biomarker is GATA1, GATA2, C/ EBP α, FLI1, FOG1, ETS1, PU.1, RUNX1 and CBF α one kind or combinations thereof.

9. according to the method any one of claim 1-7, wherein selected biomarker be BCL2, CCNA1, CD44、C/EBPα、CBFβ、CSF1、CXCL10、CXCR4、ETS1、ETS2、FLI1、FOG1、FCER1A、GATA1、GATA2、 GFI1B, HEB, IRF1, IRF8, JAG1, LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1 and TNF one kind Or combinations thereof.

10. method as claimed in one of claims 1-7, wherein selected biomarker be composing type STAT1-pS727, WT1 mutation, TET2 mutation, IDH1 mutation, IDH2 mutation, MLL reset, C/EBP α mutation, CBF β reset, PU.1 mutation, GATA1 Or 2 mutation, ERG transpositions, TLX1 be overexpressed and TLX3 activation in one kind or combinations thereof.

11. according to the method any one of claim 1-10, also including the use of independently selected from the and of claim 4,8,9 At least two biomarkers listed by any one of 10.

12. according to the method any one of claim 1-10, also including the use of independently selected from the and of claim 4,8,9 At least three kinds of biomarkers listed by any one of 10.

13. according to the method any one of claim 1-10, also including the use of independently selected from the and of claim 4,8,9 At least four biomarkers listed by any one of 10.

14. according to the method any one of claim 1-13, wherein the tumour or cancer be hematopoietic lineage tumour or Cancer.

15. according to the method for claim 14, wherein the hematopoietic lineage tumour or cancer are selected from：Acute lymphoblast Property leukaemia (ALL), acute myeloid leukaemia (AML), chronic lymphoblastic leukaemia (CLL), B cell are acute into lymph Cell leukemia (B-ALL), children B-ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute macronucleus are thin Born of the same parents' property leukaemia, Hodgkin lymphoma, NHL, Burkitt lymphoma, aids related lymphoma, Chronic Myeloid Proliferative diseases, primary central nervous system lymphoma, t cell lymphoma, hair cell leukaemia and Huppert's disease (MM), or wherein described cell is hematopoetic tumor or the precursor of cancer, such as myelodysplastic syndrome (MDS).

16. according to the method any one of claim 1-13, wherein the tumour or cancer are non-hematopoietic lineage tumours Or cancer.

17. according to the method for claim 16, wherein the tumour or cancer be breast cancer, oophoroma, carcinoma of endometrium, Squamous cell carcinoma, angiosarcoma, colon cancer, stomach and intestine tumor, metastasis tendency solid tumor, clear cell carcinoma, clear-cell carcinoma or food Road cancer.

18. according to the method any one of claim 1-17, wherein the cortex chalone for being applied to patient is selected from formula (A-1)、(A-1′)、(A-1″)、(A-2′)、(A-2″)、(A-3′)、(A-3″)、(D1′)、(D1″)、(D2′)、(D2″)、 The compound of (E1 '), (E1 "), (E2 '), (E2 "), (G1 ') or (G1 ").

19. according to the method any one of claim 1-17, wherein being applied to the cortex chalone of the patient is：

20. according to the method any one of claim 1-17, wherein being applied to the cortex chalone of the patient is Natural cortex chalone.

21. according to the method any one of claim 1-17, wherein, it is applied to the cortex chalone choosing of the patient From pharmaceutically acceptable known cortex chalone derivative.

22. according to the method any one of claim 1-21, wherein the RUNX1 is impaired is RUNX1 point mutation, is related to The chromosome translocation of RUNX1 genes or caused by causing stabilization removal or the increased mutation of degraded of RUNX1 albumen.

23. according to the method any one of claim 1-22, wherein the RUNX1 transcription factors are impaired to cause RUNX1 Gene expression under control reduces.

24. the method for the patient for targetting selection and treatment response cortex chalone treatment, it includes (i) and determines that the patient is It is no to have selected from the ER positives, VHL functions loss mutation (VHL- feminine genders), HER2 overexpressions, EGFR mutation, MET mutation, neural mother The biomarker of cytoma；EWS-FLI1, STAT1-pS727, STAT1, or ETV1, FLI1, SMC3, SMC1A, RAD21 or One kind or combinations thereof of Inactivating mutations in STAG2；And if it has, (ii) apply effective dose cortex chalone or its Pharmaceutically acceptable salt, oxide or optionally with pharmaceutically acceptable composition.

25. for predicting the patient with tumour or cancer to the method for the response treated with cortex chalone, it includes：

I. tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. determine that the expression of assessment or expression quantity are whether outside the scope of corresponding normal cell in step (ii), example Such as, higher or lower than the scope found in corresponding normal cell, or higher or lower than with the increase of the clinical Benefit of patient or Reduce a certain amount of of correlation；With

Iv. optionally with the cortex chalone or its pharmaceutically acceptable salt or oxide of effective dose optionally with its pharmacy Upper acceptable composition treatment patient.

26. for selecting the method for the patient for having response to the treatment of cortex chalone, it includes：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, with described in determination Whether patient has response to the treatment of cortex chalone, and the control sample has response including representative number to cortex chalone Patient or predictive animal model and representative number to cortex chalone without or have the patient of poor response；With

Iv. if it is determined that the patient has response to the treatment, then optionally being applied with its pharmaceutically acceptable composition has The cortex chalone or its pharmaceutically acceptable salt or oxide of effect amount.

27. according to the method any one of claim 24-26, also including the use of for determining whether patient will successfully The kit of cortex chalone treatment is responded, wherein the kit includes the multinuclear with biomarker or biomarker combinations The probe or the antibody of combination biomarker protein that thuja acid is annealed under strict conditions.

28. according to the method any one of claim 24-27, include the kit of gene selected by diagnosis, it is described to examine Disconnected kit includes primer and optionally heat-staple for being used to expand the DNA complementary with the RNA of gene specific coding Archaeal dna polymerase.

29. according to the method any one of claim 24-27, in addition to wherein every kind of primer is in the strict bar of standard Under part with the RNA of the gene code or the kit of its complementary sequence hybridization.

30. according to the method any one of claim 24-27, wherein the tumour or cancer be hematopoietic lineage tumour or Cancer.

31. according to the method for claim 30, wherein the hematopoietic lineage tumour or cancer are selected from：Acute lymphoblast Property leukaemia (ALL), B cell acute lymphoblastic leukemia (B-ALL), children B-ALL, acute myeloid leukaemia (AML), chronic lymphoblastic leukaemia (CLL), B cell acute lymphoblastic leukemia (B-ALL), children B- It is ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute megakaryoblastic leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, Burkitt lymphoma, aids related lymphoma, Chronic Myeloid proliferative diseases, Primary Central Nervous System lymphomas, t cell lymphoma, hair cell leukaemia and Huppert's disease (MM), or wherein described cell is to make The precursor of hemotoncus knurl or cancer, such as myelodysplastic syndrome (MDS).

32. according to the method any one of claim 22-27, wherein the tumour or cancer are non-hematopoietic lineage tumours Or cancer.

33. according to the method for claim 32, wherein the tumour or cancer be breast cancer, oophoroma, carcinoma of endometrium, Squamous cell carcinoma, colon cancer, stomach and intestine tumor, metastasis tendency solid tumor, clear cell carcinoma, clear-cell carcinoma or the cancer of the esophagus.

34. the method for having the tumour of response or the patient of cancer with confrontation CDK8/19 treatments for targetting selection and treatment, its Determine whether the patient there is RUNX1 approach to be damaged including (i)；If it has, the CDK8/19 that (ii) applies effective dose suppresses Agent or its pharmaceutically acceptable salt, oxide or optionally with pharmaceutically acceptable composition.

35. the method for treating the impaired tumour of RUNX1 in patient or cancer, it is included by being transcribed with producing by RUNX1 Albumen abundant up-regulation mode and dosage apply effective dose CDK8/19 inhibitor so that cell more normal, virulence are more Weak, more ripe, prevention cell growth or the mode of apoptosis cause the differentiation of the tumour or cancer.

36. for predicting the method for tumour or cancer patient to the response with CDK8/19 inhibitor for treating, it includes：

I. tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. determine that the expression of assessment or expression quantity are whether outside the scope of corresponding normal cell in step (ii), example Such as, higher or lower than the scope found in corresponding normal cell, or higher or lower than with the increase of the clinical benefit of patient or Reduce a certain amount of of correlation；With

Iv. optionally optionally pharmaceutically may be used with the cortex chalone of effective dose or its pharmaceutically acceptable salt or oxide with it Patient described in the composition treatment of receiving.

37. being suitable for the method for the tumour or cancer patient with CDK8/19 inhibitor for treating for selection, it includes：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, with described in determination Whether patient, which resists CDK8/19 treatments, response, and the control sample includes having to CDK8/19 inhibitor for representative number The patient of response or predictive animal model and representative number to CDK8/19 inhibitor without or have the trouble of poor response Person；With

Iv. if it is determined that the patient has response to the treatment, then optionally being applied with its pharmaceutically acceptable composition has The CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide of effect amount.

38. according to the method any one of claim 34-37, also including the use of for determining whether patient will successfully Respond the kit of anti-CDK8/19 treatment, wherein the kit include it is more with biomarker or biomarker combinations The probe or the antibody of combination biomarker protein that nucleotides is annealed under strict conditions.

39. according to the method any one of claim 34-38, in addition to kit, the kit includes diagnosis Gene, the primer for expanding the DNA complementary with the RNA of gene specific coding selected by impaired one group of RUNX1 approach With optional heat-staple archaeal dna polymerase.

40. according to the method any one of claim 34-38, it also includes kit, and the kit includes one group Primer, it for each gene of the impaired selected genome of diagnosis RUNX1 approach by being used to expand and the gene specific The DNA complementary RNA of coding primer composition, wherein RNA of every kind of primer under standard stringent condition with the gene code Or its complementary sequence hybridization.

41. according to the method any one of claim 34-40, wherein selected biomarker be GATA1, GATA2, C/EBP α, FLI1, FOG1, ETS1, PU.1 and CBF α one kind or combinations thereof.

42. according to any one of claim 34-40 method, wherein selected biomarker be BCL2, CCNA1, CD44, C/EBPα、CBFβ、CSF1、CXCL10、CXCR4、ETS1、ETS2、FLI1、FOG1、FCER1A、GATA1、GATA2、GFI1B、 HEB, IRF1, IRF8, JAG1, LMO2, LTB, NFE2, NOTCH2, PU.1, SLA, SOCS1, TAL1 and TNF one kind or they Combination.

43. according to any one of claim 34-40 method, wherein selected biomarker is composing type STAT1- PS727, WT1 mutation, TET2 mutation, IDH1 mutation, IDH2 mutation, MLL are reset, C/EBP α mutation, CBF β are reset, PU.1 dashes forward One kind or combinations thereof in change, the mutation of GATA1 or 2, ERG transpositions, TLX1 overexpressions and TLX3 activation.

44. according to the method any one of claim 34-40, also including the use of independently selected from claim 37,41 With at least two biomarkers listed in 42 any one.

45. according to the method any one of claim 34-43, also including the use of independently selected from claim 37,41 With any one of 42 in listed at least three kinds of biomarkers.

46. according to the method any one of claim 34-43, also including the use of independently selected from claim 37,41 With any one of 42 in listed at least four biomarkers.

47. according to the method any one of claim 34-46, also including the use of for determining whether patient will successfully The kit of CDK8/19 treatments is responded, wherein the kit includes the multinuclear with biomarker or biomarker combinations The probe or the antibody of combination biomarker protein that thuja acid is annealed under strict conditions.

48. for predicting the patient with tumour or cancer to the method for the response with CDK8/19 inhibitor for treating, it includes：

I. the tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. determine whether the expression of assessment or expression quantity are higher or lower than in corresponding normal cell in step (ii) It was found that scope, for example, being increased or decreased higher or lower than the clinical benefit to patient related a certain amount of；With

Iv. optionally with the CDK8/19 inhibitor of effective dose or its pharmaceutically acceptable salt or oxide optionally with its medicine Patient described in acceptable composition treatment on.

49. for selecting to having the tumour of response or the method for cancer patient with CDK8/19 inhibitor for treating, it includes：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, with described in determination Whether patient has response to the treatment of cortex chalone, and the control sample includes having to CDK8/19 inhibitor for representative number The patient of response or predictive animal model and representative number to CDK8/19 inhibitor without or have the trouble of poor response Person；With

Iv. if it is determined that the patient has response to the treatment, then optionally being applied with its pharmaceutically acceptable composition has The CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide of effect amount.

50. according to the method any one of claim 48-49, include the kit of gene selected by diagnosis, the examination Agent box includes the primer for being used for expanding the DNA complementary with the RNA of gene specific coding and optional heat-staple DNA gathers Synthase.

51. according to the method any one of claim 48-49, in addition to kit, the kit draws comprising one group Thing, it is by the primer for the DNA for being used to expand the RNA encoded with the gene specific complementations for each gene of selected genome Composition, wherein RNA or its complementary sequence hybridization of every kind of primer under standard stringent condition with the gene code.

52. according to the method any one of claim 48-51, wherein the tumour or cancer be hematopoietic lineage tumour or Cancer.

53. method according to claim 52, wherein the hematopoietic lineage tumour or cancer are selected from：Acute lymphoblast Property leukaemia (ALL), acute myeloid leukaemia (AML), chronic lymphoblastic leukaemia (CLL), B cell acute lymphoblastic it is female Cell leukemia (B-ALL), children B-ALL, chronic myelogenous leukemia, acute monocytic leukemia, acute macronucleus are thin Born of the same parents' property leukaemia, Hodgkin lymphoma, NHL, Burkitt lymphoma, aids related lymphoma, Chronic Myeloid Proliferative diseases, primary central nervous system lymphoma, t cell lymphoma, hair cell leukaemia and Huppert's disease (MM), or wherein described cell is hematopoetic tumor or the precursor such as myelodysplastic syndrome (MDS) of cancer.

54. according to the method any one of claim 48-51, wherein the tumour or cancer are non-hematopoietic lineage tumours Or cancer.

55. method according to claim 54, wherein the tumour or cancer be breast cancer, oophoroma, carcinoma of endometrium, Squamous cell carcinoma, angiosarcoma, colon cancer, stomach and intestine tumor, metastasis tendency solid tumor, clear cell carcinoma, clear-cell carcinoma or food Pipe cancer.

56. treat the patient according to the method any one of claim 1-55, in addition to the second activating agent.

57. the patient is treated according to the method any one of claim 1-55, in addition to the second activating agent, wherein Second activating agent is selected from BET inhibitor, PI3K inhibitor, Raf inhibitor, BTK inhibitor, Bcl-2 inhibitor, CDK7 suppressions Preparation, mek inhibitor or Syk inhibitor.

58. the patient is treated according to the method any one of claim 1-55, in addition to the second activating agent, wherein Second activating agent is selected from nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech/ Teva), AMP-244 (Amplimmune/GSK), BMS-936559 (BMS) and MEDI4736 (Roche/Genentech) PD- 1 inhibitor.

59. according to the method any one of claim 1-55, in addition to described at least one other activating agents treatment Patient, wherein second activating agent be selected from JQ1, I-BET151 (also known as GSK1210151A), I-BET762 (also known as GSK525762), OTX-015 (also known as MK-8268, IUPAC 4- (4- chlorphenyls)-N- (4- hydroxy phenyls) -2,3,9- front threes Base -) 6H- thienos [3,2-f] [1,2,4] triazol [4,3-a] [1,4] diaza- 6- acetamides, TEN-010, CPI- 203rd, CPI-0610, RVX-208 and LY294002 BET inhibitor.

60. the patient is treated according to the method any one of claim 1-55, in addition to the second activating agent, wherein Other described activating agents are immunomodulators.

61. according to the method any one of claim 1-55, wherein other described activating agents are anti-PD1 antibody.

62. according to the method any one of claim 1-55, wherein other described activating agents are anti-CTLA-4 compounds, Such as ipilimumab (Yervoy) or tremelimumab.

63. according to the method any one of claim 1-55, wherein the patient suffers from tumour.

64. according to the method any one of claim 1-55, wherein the patient suffers from cancer.

65. the as above kit described in any embodiment.

66. the combination dosage forms of cortex chalone and other at least one activating agents, it using claim 1-10 method with being carried out The diagnosticum of patient's selection is used in combination.

67. cortex chalone or its pharmaceutically acceptable salt or oxide are optionally used for its pharmaceutically acceptable composition Targeting selection and treatment have the tumour of response or the method for cancer patient to the treatment of cortex chalone, and wherein methods described includes (i) Determine whether the patient there is RUNX1 approach to be damaged；If it has, (ii) applies the compound of effective dose.

68. cortex chalone is used for the method for treating the impaired tumour of RUNX1 in patient or cancer, wherein methods described is included with production Give birth to the mode of the abundant up-regulation of the albumen generally by RUNX1 transcriptions and dosage applies the compound of effective dose, so that cell is corrected Often, the differentiation that virulence is weaker, the more ripe or mode of prevention growth or apoptosis causes the tumour or cancer.

69. cortex chalone or its pharmaceutically acceptable salt or oxide are optionally used for its pharmaceutically acceptable composition The method of tumour or cancer patient to the response with the compounds for treating is predicted, wherein methods described includes：

I. the tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. determine that the expression of assessment or expression quantity are whether outside the scope of corresponding normal cell in step (ii), example Such as, higher or lower than the scope found in corresponding normal cell, or higher or lower than with the increase of the clinical Benefit of patient or Reduce a certain amount of of correlation；With

Iv. the patient optionally described in the compounds for treating of effective dose.

70. cortex chalone or its pharmaceutically acceptable salt or oxide are optionally used for its pharmaceutically acceptable composition The method that selection is suitable for the tumour or cancer patient treated with cortex chalone, wherein methods described include：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, to determine patient Whether response is had to the treatment of cortex chalone, the control sample includes the patient for having response to cortex chalone of representative number Predictive animal model and representative number to cortex chalone without or have the patient of poor response；With

Iv. if it is determined that the patient has response to the treatment, then using the compound of effective dose.

71. cortex chalone or its pharmaceutically acceptable salt or oxide are optionally used for its pharmaceutically acceptable composition Targeting selection and the method for treating the patient for having response to the treatment of cortex chalone, wherein methods described determine that patient is including (i) It is no to have selected from the ER positives, VHL functions loss mutation (VHL- feminine genders), HER2 overexpressions, EGFR mutation, MET mutation, neural mother The biomarker of cytoma；EWS-FLI1, STAT1-pS727, STAT1, or ETV1, FLI1, SMC3, SMC1A, RAD21 or One kind or combinations thereof of Inactivating mutations in STAG2；And if it has, (ii) applies the compound of effective dose.

72. cortex chalone or its pharmaceutically acceptable salt or oxide are optionally used for its pharmaceutically acceptable composition Patient of the prediction with tumour or cancer includes to the method for the response treated with cortex chalone, wherein methods described：

I. the tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. determine that the expression of assessment or expression quantity are whether outside the scope of corresponding normal cell in step (ii), example Such as, higher or lower than the scope found in corresponding normal cell, or higher or lower than with the increase of the clinical Benefit of patient or Reduce a certain amount of of correlation；With

Iv. the patient optionally described in the compounds for treating of effective dose.

73. cortex chalone or its pharmaceutically acceptable salt or oxide are optionally used for its pharmaceutically acceptable composition By to there is the method for the patient of response with the treatment of cortex chalone, wherein methods described includes for selection：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, to determine patient Whether response is had to the treatment of cortex chalone, the control sample includes the patient for having response to cortex chalone of representative number Predictive animal model and representative number to cortex chalone without or have the patient of poor response；With

Iv. if it is determined that the patient has response to the treatment, then using the compound of effective dose.

74.CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide are optionally with its pharmaceutically acceptable combination Thing is used to target selection and treatment resists CDK8/19 treatments and has the tumour of response or the method for cancer patient, wherein methods described Determine whether patient there is RUNX1 approach to be damaged including (i)；If it has, (ii) applies the compound of effective dose.

75.CDK8/19 inhibitor is used for the method for treating the impaired tumour of RUNX1 in patient or cancer, wherein methods described bag Include in a manner of producing by the abundant up-regulation of the albumen of RUNX1 transcriptions and dosage applies the compound of effective dose, so that carefully Born of the same parents' more normal, virulence be weaker, it is more ripe, prevent cell growth or the mode of apoptosis from causing the differentiation of the tumour or cancer.

76.CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide are optionally with its pharmaceutically acceptable combination Thing is used to predict method of the patient with tumour or cancer to the response with CDK8/19 inhibitor for treating, wherein methods described Including：

I. tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. determine that the expression of assessment or expression quantity are whether outside the scope of corresponding normal cell in step (ii), example Such as, higher or lower than the scope found in corresponding normal cell, or higher or lower than with the increase of the clinical Benefit of patient or Reduce a certain amount of of correlation；With

Iv. the patient optionally described in the compounds for treating of effective dose.

77.CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide are optionally with its pharmaceutically acceptable combination Thing is used to select the method for being suitable for tumour or cancer patient with CDK8/19 inhibitor for treating, and wherein methods described includes：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ACSL1、ADORA2B、ADRB1、AMPD3、ARRDC4、BCL2、 BCL2A1、CBFβ、CCNA1、CD244、CD44、CDC42EP3、C/EBPα、CECR6、CFLAR、CISH、CSF1、CXCL10、 CXCR4、CYTIP、DUSP10、E2F8、EMB、EMR2、ETS1、ETS2、FAM107B、FAM46A、FCER1A、FCGR1B、FLI1、 FOG1、FOSL2、GAB2、GAS7、GATA1、GATA2、GFI1B、GMPR、GPR18、GPR183、HBBP1、HEB、HLX、 HMGCS1、IGFBP4、IGFBP5、IL17RA、IL1RAP、IPCEF1、IRF1、IRF8、ITGA6、JAG1、LCP2、LDLR、 LIMA1、LMO2、LRRC33、LTB、MBP、MICAL2、MYCN、MYO1G、NFE2、NOTCH2、NRP1、P2RY2、PAG1、 PLAC8、PLEK、PLXNC1、PMP22、PTPRE、PU.1、PXK、RAB27A、RASA3、RGS16、RHOH、RNF24、RXRA、 SELPLG、SLA、SLC7A11、SLC7A5、SOCS1、ST3GAL4、STK17B、TAL1、TIMP3、TMEM104、TNF、 TSC22D1, TSC22D3, ZBTB16 and ZCCHC5；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, to determine patient Whether resisting CDK8/19 treatments has response, and the control sample has response including representative number to CDK8/19 inhibitor Patient or predictive animal model and representative number to CDK8/19 inhibitor without or have the patient of poor response；With

Iv. if it is determined that the patient has response to the treatment, then using the compound of effective dose.

78.CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide are optionally with its pharmaceutically acceptable combination Thing is used to predict method of the patient with tumour or cancer to the response with CDK8/19 inhibitor for treating, wherein methods described Including：

I. the tumour or cancer specimen are obtained from the patient；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from patient are determined, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. determine whether the expression of assessment or expression quantity are higher or lower than in corresponding normal cell in step (ii) It was found that scope, for example, related a certain amount of higher or lower than increasing or decreasing for the clinical Benefit to patient；With

Iv. the patient optionally described in the compounds for treating of effective dose.

79.CDK8/19 inhibitor or its pharmaceutically acceptable salt or oxide are optionally with its pharmaceutically acceptable combination Thing is used to select by having the tumour of response or the method for cancer patient, wherein methods described bag with CDK8/19 inhibitor for treating Include：

I. the tumour or cancer specimen of the patient is obtained；

Ii. the expression or expression quantity of one or more biomarkers in the biological sample from the patient are detected, Wherein described biomarker is selected from the group consisted of：ER is positive, VHL functions loss mutation (VHL- is negative), HER2 mistakes Expression, EGFR mutation, MET mutation, the biomarker of neuroblastoma；EWS-FLI1, STAT1-pS727, STAT1, or Inactivating mutations in ETV1, FLI1, SMC3, SMC1A, RAD21 or STAG2；

Iii. the expression determined in step (ii) is compared with the mutually isogenic expression in control sample, with described in determination Whether patient has response to the treatment of cortex chalone, and the control sample includes having to CDK8/19 inhibitor for representative number The patient of response or predictive animal model and representative number to CDK8/19 inhibitor without or have the trouble of poor response Person；With

Iv. if it is determined that the patient has response to the treatment, then using the compound of effective dose.